DNA vaccine against feline immunodeficiency virus

ABSTRACT

The present invention is directed to vaccine compositions that can be used to protect cats against feline immunodeficiency virus. More particularly, the present invention relates to polynucleotide molecules that can be used as vaccine components against feline immunodeficiency virus.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This patent application is a divisional patent application ofU.S. Ser. No. 09/593,580, filed Jun. 14, 2000, which claims the benefitof priority of U.S. Provisional Serial No. 60/138,999, filed Jun. 14,1999, all of which are incorporated herein by reference.

1. FIELD OF THE INVENTION

[0002] The present invention is in the field of animal health, and isdirected to vaccine compositions and diagnostics for disease. Moreparticularly, the present invention relates to polynucleotide moleculesthat can be used as vaccine components against feline immunodeficiencyvirus.

2. BACKGROUND OF THE INVENTION

[0003] Feline immunodeficiency virus (FIV) infection in cats results ina disease syndrome similar to that caused in humans by humanimmunodeficiency virus-1 (HIV-1) infection. After infection of cats byFIV, disease progression begins with a transient acute phase illness (8to 10 weeks), followed by a prolonged asymptomatic phase varying fromweeks to years, and a terminal symptomatic phase (Ishida and Tomoda,1990, Jpn. J. Vet Sci. 52:645-648; English et al., 1994, J. Infect. Dis.170: 543-552). Similar to HIV-1 disease progression (Graziosi et al.,1993, Proc. Natl. Acad. Sci. 90:6405-6409; Baumberger et al., 1993, AIDS7:S59-S64; Wei et al., 1995, Nature 373:117-122), FIV RNA load in plasmahas been demonstrated to correlate with disease stage, and can predictdisease progression in accelerated FIV infection (Diehl et al., 1995, J.Virol. 69:2328-2332; Diehl et al., 1996, J. Virol. 70:2503-2507).

[0004] Based on the genetic diversity of the ENV protein of FIV,especially the V3 region, five FIV subtypes have been proposed: subtypesA and B, mainly in North America, Europe and Japan; subtype C in BritishColumbia and Taiwan; subtype D in Japan; and subtype E in Argentina(Sodora et al., 1994, J. Virol. 68:2230-2238; Kakinuma et al., 1995, J.Virol. 69:3639-3646; and Pecoraro et al., 1996, J. Gen. Virol.77:2031-2035).

[0005] Similar to other lentiviruses, such as HIV-1, the FIV genomecontains three large open reading frames, ie., GAG (group antigens), ENV(envelope), and POL (polymerase), and three small open reading framesencoding regulatory (i.e., non-structural) proteins, i.e., Rev(regulator of expression of virion protein), Vif (virion infectivityfactor) and ORF2 (open reading frame 2). The provirus contains two longterminal repeats (LTR), one at each end of the genome (Talbott et al.,1989, Proc. Natl. Acad. Sci. USA 86:5743-5747; Olmsted et al., 1989,Proc. Natl. Acad. Sci. USA 86:8088-8092). GAG is a precursor polyproteinthat is processed into three mature virion structural proteins, i.e.,the matrix (MA), capsid (CA) and nucleocapsid (NC) proteins. ENV is aprecursor protein that is processed into two envelope structuralproteins, i.e., the surface (SU) and transmembrane (TM) proteins. POLencodes four enzymatic (i.e., non-structural) proteins, i.e., protease(PR), reverse transcriptase (RT), deoxyuridine triphosphatase (DU) andintegrase (IN).

[0006] The mechanism by which protective immunity against FIV infectioncan be achieved remains poorly understood. It has been reported by somegroups that virus neutralizing (VN) antibodies appear to play a majorrole in the observed protection (Yamamoto et al., 1991, AIDS Res. Hum.Retrovir. 7:911-922; Hosie et al., 1995, J. Virol. 69:1253-1255).Consistent with those observations was the finding that cats whopassively received antibodies from vaccinated or infected cats wereprotected from homologous challenge (Hohdatsu et al., 1993, J. Virol.67:2344-2348; Pu et al., 1995, AIDS 9:235-242).

[0007] By contrast, convincing data also indicates that the levels ofantibodies, or even VN antibodies, do not correlate with protection. Ithas been reported that cats were protected against homologous challengein the absence of detectable VN antibodies (Verschoor et al., 1995, Vet.Immunol. Immunopathol. 46:139-149; Matteucci et al., 1996, J. Virol.70:617-622). In addition, other vaccinated cats failed to be protectedin the presence of significant VN antibodies (Huisman et al., 1998,Vaccine 16:181-187; Flynn et al., 1997 J. Virol. 71:7586-7592; Tijhaaret al., 1997, Vaccine 15:587-596; Osterhaus et al., 1996, AIDS Res. Hum.Retrovir. 12:437-441; Verschoor et al., 1996, Vaccine 14:285-289; Rigbyet al., 1996, Vaccine 14:1095-1102; Lutz et al., 1995, Vet. Immunol.Immunopathol. 46:103-113; Flynn et al., 1995, Immunol. 85:171-175; Goninet al., 1995, Vet. Microbiol. 45:393-401). This discrepancy appears toresult, at least partially, from the different cell systems and virusisolates used in the VN assays. It has recently become evident thatfresh isolates of FIV obtained from naturally infected cats are muchless sensitive to VN antibodies than laboratory viruses adapted togrowth in tissue culture (Baldinotti et al., 1994, J. Virol. 68:4572-4579). It has also been found that the same antibodies whichneutralized FIV infection in Crandell Feline Kidney (CRFK) cells failedto neutralize FIV infection in primary feline thymocytes (Huisman etal., 1998, above). These data indicate that the VN antibodies detectedin vitro may not play any role in protective immunity in vivo.

[0008] In a few limited reports, cell-mediated immunity was investigatedfollowing vaccination. In one report, it was found that cellularimmunity, especially ENV-specific CTL responses, played a major role inprotecting cats vaccinated with whole inactivated virus (Flynn et al.,1996, J. Immunol. 157:3658-3665; Flynn et al., 1995, AIDS Res. Hum.Retrovir. 11:1107-1113). It was also reported that long-term protectionwas more closely correlated with the induction of ENV-specific cytotoxicT-cell activity (Hosie and Flynn, 1996, J. Virol. 70:7561-7568).

[0009] It appears that both humoral and cellular immunity are involvedin achieving protective immunity in the acute phase after challenge, butfor long-term protection, cell-mediated immunity appears to be moreimportant. However, the question still remains which viral protein(s) orsubunit(s) or combinations are capable of inducing protective immuneresponses. In one report, although both cell-mediated and humoral immuneresponses were induced in cats vaccinated with a multi-epitopic peptidewithin the ENV protein, vaccination did not confer protection againsthomologous challenge (Flynn et al., 1997, above).

[0010] As in HIV-1, an observation that complicates the development ofan effective FIV vaccine is the enhancement of infection that has beenobserved in cats immunized with certain vaccines. Such enhancement ofinfection has been observed in a number of FIV vaccine trials in whicheither recombinant subunit vaccines, synthetic vaccines, wholeinactivated virus vaccines or fixed, infected cell vaccines were used tovaccinate cats (Osterhaus et al., 1996, above; Siebelink et al., 1995,J. Virol. 69:3704-3711; Lombardi et al., 1994, J. Virol. 68:8374-8379;Hosie et al., 1992, Vet. Immunol. Immunopathol. 35:191-197; Huisman etal, 1998, above). For example, in an ENV subunit vaccine trial,enhancement of infection occurred despite anti-ENV and VN antibodyproduction, and this enhancement could be transferred to naïve cats viaplasma pools from the vaccinated animals, indicating that theenhancement was probably mediated by specific antibodies (Siebelink etal., 1995, above).

[0011] It appears that antibodies against ENV tend to enhance infectionmore readily than antibodies against GAG protein. However, the mechanismby which antibodies enhance FIV infection remains poorly understood. InHIV-1, antibody-dependent enhancement requires that the target cellsexpress either the immunoglobulin Fc receptor (FcR), or complementreceptors (CRs). The enhancement is a biphasic response based on serumdilution; that is, at higher antibody concentrations, viralneutralization is observed, whereas enhancement is seen at lowerantibody concentrations (Mascola et al., 1993, AIDS Res. Hum. Retrovir.9:1175-1184). The enhanced infectivity may interfere with the inductionof protective immunity in FIV , which may partially explain the reasonwhy a large number of FIV vaccination experiments in which ENV proteinor its subunits were used as vaccines were unsuccessful. Therefore, therational development of vaccines against lentiviruses, including FIV andHIV-1, requires the careful assessment and selection of vaccineimmunogens.

[0012] Since the discovery of FIV, many attempts have been made todevelop a safe and effective FIV vaccine. Three different groups haveattempted to vaccinate cats with fixed virus-infected cells; however,conflicting results were obtained from these vaccination trials. Thefirst group found that all the cats vaccinated with fixed FIV-infectedcells were protected from challenge with plasma obtained from catsinfected with the homologous virus, despite the fact that no VNantibodies were detected after vaccination (Matteucci et al., 1996,above). The protection conferred by this vaccine, however, wasrelatively short-lived and difficult to boost (Matteucci et al., 1997,J. Virol. 71:8368-8376). Similar results were reported by the secondgroup describing protection against homologous, but not heterologous,FIV challenge up to 12 weeks post-challenge (Bishop et al., 1996,Vaccine 14:1243-1250). However, when cats were monitored up to week 50post-challenge, a loss of protection against the homologous virus wasobserved. Also, protection could not be correlated with the levels ofantibody to p24 capsid protein or VN titers. In contrast to thesefindings, the third group reported no protection when ten cats werevaccinated with a fixed FIV-infected cell vaccine. Eight of the catsbecame viraemic 5 weeks post-challenge, although significant VNantibodies were detected at the time of challenge (Verschoor et al.,1995, above).

[0013] Another type of conventional FIV vaccine that has been tested iswhole, inactivated virus. The first successful whole-inactivated FIVvaccine was reported by Yamamoto's group, which observed greater than90% protection against homologous challenge (Yamamoto et al., 1991, AIDSRes. Hum. Retrovir. 7:911-922), and slight protection againstheterologous challenge (Yamamoto et al., 1993, J. Virol. 67:601-605).Both humoral and cellular immunity against FIV were induced and highlevels of anti-ENV, anti-core, and VN antibodies were observed in thevaccinated cats. Recent studies have indicated that both virus-specifichumoral immunity, especially VN antibodies, and cellular immunity,especially the ENV-specific CTL responses, play a role in the protectioninduced in cats vaccinated with whole, inactivated virus (Hosie andFlynn, 1996, above; Flynn et al., 1996, above; Hosie et al., 1995,above; Elyar et al., 1997, Vaccine 15:1437-1444). However, in contrastto the studies described above, vaccination of cats with whole,inactivated FIV incorporated into immune stimulating complexes (ISCOMs)failed to protect against homologous challenge (Hosie et al., 1992,above).

[0014] Another approach for FIV vaccine development that has beenextensively investigated recently is recombinant vaccines. A number ofFIV subunit vaccines have been tested, including those containingrecombinant core protein, synthetic V3, or multi-epitopic peptides,glycosylated or unglycosylated recombinant ENV protein, and variousvector-based systems (Elyar et al., 1997, above). Unfortunately,although significant levels of antibodies were generally induced by suchvaccinations, all attempts failed to protect vaccinated cats againsthomologous challenge (Huisman et al., 1998, above; Flynn et al., 1997,above; Tijhaar et al., 1997, above; Osterhaus et al., 1996, above;Verschoor et al., 1996, above; Rigby et al., 1996, above; Lutz et al.,1995, above; Flynn et al., 1995, Immunol. 85:171-175; Gonin et al.,1995, above).

[0015] Recently, a DNA vaccine was tested for FIV. Cats vaccinated withplasmid DNA carrying FIV structural genes, including ENV and p10 gene(i.e., the NC protein of FIV), exhibited strong humoral immuneresponses. However, none of the vaccinated cats were protected fromhomologous challenge (Cuisinier etal., 1997, Vaccine 15: 1085-1094).

[0016] In addition, WO 98/03660 describes various formulae for felinepolynucleotide vaccines including against FIV, but only mentions the useof ENV polyprotein and GAG/PRO polyprotein genes, and does not describethe use of other FIV genes, or substituent genes from the particularpolyprotein genes, nor does it provide any data showing efficacy of anyparticular FIV vaccine.

3. SUMMARY OF THE INVENTION

[0017] The present invention provides a vaccine composition againstfeline immunodeficiency virus (FIV), comprising an immunologicallyeffective amount of a polynucleotide molecule comprising a nucleotidesequence selected from a portion of the genome of an FIV strain, or anucleotide sequence which is a degenerate variant thereof; and aveterinarily acceptable carrier. The FIV strain can be any strain ofFIV, but is preferably strain FIV-141 having a genomic RNA sequencecorresponding to the DNA sequence shown in SEQ ID NO:1 from nt 1 to nt9464.

[0018] In a preferred embodiment, the polynucleotide molecule of thevaccine composition comprises a nucleotide sequence encoding one or moreof a structural or non-structural protein from an FIV strain, or acombination thereof. The structural protein is selected from the groupconsisting of a GAG protein and an ENV protein. The non-structuralprotein is selected from the group consisting of a POL protein and aregulatory protein. The GAG protein is selected from the groupconsisting of the GAG polyprotein and its substituent proteins, i.e.,MA, CA and NC. The ENV protein is selected from the group consisting ofthe ENV polyprotein and its substituent proteins, i.e., SU and TM. ThePOL protein is selected from the group consisting of the POL polyproteinand its substituent proteins, i.e., PR, RT, DU and IN. The regulatoryprotein is selected from the group consisting of Rev, Vif and ORF2.

[0019] The polynucleotide molecule of the vaccine composition mayalternatively or additionally comprise a nucleotide sequence consistingof a substantial portion of any of the aforementioned nucleotidesequences. In a preferred embodiment, the substantial portion of thenucleotide sequence encodes an epitope of an FIV protein.

[0020] In a preferred embodiment, the vaccine composition of the presentinvention comprises a polynucleotide molecule comprising a nucleotidesequence encoding an FIV protein selected from the group consisting ofGAG, MA, CA, NC, ENV, SU, TM, DU and PR.

[0021] In a more preferred embodiment, the vaccine composition of thepresent invention is a combination vaccine, which comprises one or morepolynucleotide molecules having nucleotide sequences encoding acombination of FIV proteins. In a preferred embodiment, the one or morepolynucleotide molecules of the vaccine composition comprise nucleotidesequences encoding at least two different FIV proteins selected from FIVstructural and FIV non-structural proteins, provided that when the oneor more polynucleotide molecules encode the ENV and NC proteins fromFIV, they also encode at least one, preferably at least two, and mostpreferably at least three other FIV structural or non-structuralproteins.

[0022] In a further preferred embodiment, the one or more polynucleotidemolecules of the vaccine composition comprise nucleotide sequencesencoding at least two different GAG proteins from FIV.

[0023] In a further preferred embodiment, the one or more polynucleotidemolecules of the vaccine composition comprise nucleotide sequencesencoding at least one FIV structural protein and at least one FIVnon-structural protein.

[0024] In a further preferred embodiment, the one or more polynucleotidemolecules of the vaccine composition comprise nucleotide sequencesencoding at least three different FIV proteins selected from among theFIV structural and FIV non-structural proteins, i.e., the proteins canbe either all structural proteins or all non-structural proteins, or acombination of structural and non-structural proteins. In a furtherpreferred embodiment, the one or more polynucleotide molecules of thevaccine composition comprise nucleotide sequences encoding at least fourdifferent FIV proteins selected from among the FIV structural and FIVnon-structural proteins. In a further preferred embodiment, the one ormore polynucleotide molecules of the vaccine composition comprisenucleotide sequences encoding at least five different FIV proteinsselected from among the FIV structural and FIV non-structural proteins.In a further preferred embodiment, the one or more polynucleotidemolecules of the vaccine composition comprise nucleotide sequencesencoding at least six different FIV proteins selected from among the FIVstructural and FIV non-structural proteins. In a further preferredembodiment, the one or more polynucleotide molecules of the vaccinecomposition comprise nucleotide sequences encoding at least sevendifferent FIV proteins selected from among the FIV structural and FIVnon-structural proteins.

[0025] In a further preferred embodiment, the one or mor polynucleotidemolecules of the vaccine composition comprise a nucleotide sequenceencoding at least one FIV structural protein and a nucleotide sequenceencoding at least one FIV regulatory protein. In a further preferredembodiment, the one or more polynucleotide molecules of the vaccinecomposition comprise a nucleotide sequence encoding at least one FIV POLprotein and a nucleotide sequence encoding at least one FIV regulatorygene. In a further preferred embodiment, the one or more polynucleotidemolecules of the vaccine composition comprise a nucleotide sequenceencoding at least one FIV structural protein, a nucleotide sequenceencoding at least one FIV POL protein, and a nucleotide sequenceencoding at least one FIV regulatory protein.

[0026] In a further preferred embodiment, when the one or morepolynucleotide molecules of the vaccine composition comprise nucleotidesequences encoding a GAG protein, PR protein or ENV protein from FIV, ora combination thereof, one or more nucleotide sequences encoding atleast one, more preferably at least two, and most preferably at leastthree other FIV proteins are present.

[0027] In a particularly preferred embodiment, the vaccine compositionof the present invention comprises one or more polynucleotide moleculescomprising nucleotide sequences encoding a combination of FIV proteins,which combination is selected from the group consisting of GAG/MA/CA/NC;GAG/ENV; GAG/MA/CA/NC/ENV/SU/TM; MA/CA/NC; GAG/MA/NC/DU/PR; andMA/CA/NC/SU/TM. When the vaccine composition of the present invention isa combination vaccine, the nucleotide sequences encoding the various FIVproteins or polypeptides can be on the same polynucleotide molecule, ondifferent polynucleotide molecules, or a combination thereof.

[0028] The polynucleotide molecule of the vaccine composition can eitherbe a DNA or RNA molecule, although DNA is preferred. The polynucleotidemolecule of the vaccine composition is preferably administered as partof an expression vector construct, such as a plasmid or a viral vector.

[0029] The present invention further provides a method of preparing avaccine composition against FIV, comprising combining an immunologicallyeffective amount of any one or more of the aforementioned polynucleotidemolecules, or any one or more expression vectors comprising suchpolynucleotide molecules, with a veterinarily acceptable carrier in aform suitable for administration to cats. In a non-limiting embodiment,a veterinarily acceptable carrier is selected from standard aqueous orpartially aqueous solutions, such as sterile saline or PBS, or cationiclipid preparations, or gold microparticles onto which the one or morepolynucleotide molecules or expression vectors of the vaccinecomposition can be coated and administered to an animal for vaccinedelivery. The vaccine composition can further comprise a supplementalcomponent such as, e.g., an immunomodulatory agent, which can be anadjuvant, or a cytokine, or a polynucleotide molecule having anucleotide sequence encoding a cytokine; or an agent which facilitatescellular uptake by the vaccinated animal of the polynucleotide moleculeor expression vector; or a combination thereof.

[0030] The present invention further provides a method of vaccinating acat against FIV, comprising administering to the cat a vaccinecomposition of the present invention. In a preferred though non-limitingembodiment, the vaccine composition of the present invention isadministered to a cat either by intramuscular or intradermal injection,or orally, intranasally, or by use of a gene gun.

[0031] The present invention further provides a kit for vaccinating acat against FIV, comprising a first container comprising animmunologically effective amount of any one or more of theaforementioned polynucleotide molecules or expression vectors of thepresent invention. In a non-limiting embodiment, the one or morepolynucleotide molecule or expression vectors are stored in the firstcontainer in lyophilized form. The kit may optionally further comprise asecond container comprising a sterile diluent useful to dilute orrehydrate the polynucleotide molecules or expression vectors in thefirst container for administration to a cat.

[0032] The present invention further provides an isolated antibody thatbinds specifically to an FIV protein, which antibody is produced in amammal in response to administration of a polynucleotide molecule havinga nucleotide sequence encoding the FIV protein or an epitope thereof,such as, e.g., a polynucleotide molecule or expression vector as presentin the vaccine composition of the present invention.

[0033] The present invention further provides a vaccine compositionagainst FIV, comprising an immunologically effective amount of a GAGprotein, POL protein, ENV protein, regulatory protein, or a combinationthereof, from an FIV strain. The FIV strain can be any strain of FIV,but is preferably strain FIV-141. The GAG protein is preferably selectedfrom the group consisting of the GAG polyprotein and its substituentproteins, i.e., MA, CA and NC. The POL protein is preferably selectedfrom the group consisting of the POL polyprotein and its substituentproteins, i.e., PR, RT, DU and IN. The ENV protein is preferablyselected from the group consisting of the ENV polyprotein and itssubstituent proteins, i.e., SU and TM. The regulatory protein isselected from the group consisting of Rev, Vif and ORF2.

[0034] In a more preferred embodiment, the vaccine composition of thepresent invention comprises a combination of FIV proteins. In apreferred embodiment, the proteins of the vaccine composition compriseat least two different FIV proteins selected from among the FIVstructural and FIV non-structural proteins. In a further preferredembodiment, the proteins of the vaccine composition comprise at leasttwo different GAG proteins from FIV. In a further preferred embodiment,the proteins of the vaccine composition comprise at least one FIVstructural protein and at least one FIV non-structural protein. In afurther preferred embodiment, the proteins of the vaccine compositioncomprise at least three different FIV proteins selected from among theFIV structural and FIV non-structural proteins. In a further preferredembodiment, the proteins of the vaccine composition comprise at leastfour different FIV proteins selected from among the FIV structural andFIV non-structural proteins. In a further preferred embodiment, theproteins of the vaccine composition comprise at least five different FIVproteins selected from among the FIV structural and FIV non-structuralproteins. In a further preferred embodiment, the proteins of the vaccinecomposition comprise at least six different FIV proteins selected fromamong the FIV structural and FIV non-structural proteins. In a furtherpreferred embodiment, the proteins of the vaccine composition compriseat least seven different FIV proteins selected from among the FIVstructural and FIV non-structural proteins.

[0035] In a particularly preferred embodiment, the combination of FIVproteins is selected from the group consisting of GAG/MA/CA/NC; GAG/ENV;GAG/MA/CA/NC/ENV/SU/TM; MA/CA/NC; GAG/MA/NC/DU/PR; and MA/CA/NC/SU/TM.

[0036] Alternatively or additionally, the vaccine composition maycomprise one or more polypeptides, one or more of which is a substantialportion of an FIV protein. In a preferred embodiment, the substantialportion of the FIV protein comprises an epitope of an FIV protein.

[0037] The vaccine composition of the present invention mayalternatively comprise an immunologically effective amount of any one ormore of the aforementioned polynucleotide molecules or expressionvectors in combination with any one or more of the aforementionedproteins or polypeptides.

[0038] The present invention further provides a method of preparing avaccine composition against FIV, comprising combining an immunologicallyeffective amount of any one or more of the aforementioned proteins orpolypeptides with a veterinarily acceptable carrier in a form suitablefor administration to cats. The vaccine composition can further comprisea supplemental component such as, e.g., an immunomodulatory agent, whichcan be an adjuvant, or a cytokine, or a polynucleotide molecule having anucleotide sequence encoding a cytokine, or a combination thereof.

[0039] The present invention further provides a method of vaccinating acat against FIV, comprising administering to the cat a vaccinecomposition comprising an immunologically effective amount of any one ormore of the aforementioned proteins or polypeptides.

[0040] The present invention further comprises oligonucleotide moleculesthat can be used as primers to specifically amplify particular FIV genesor other FIV-related polynucleotide molecules, and as diagnostic probesto detect the present of an FIV-related polynucleotide molecule in afluid or tissue sample collected from an animal infected with FIV. In apreferred embodiment, such oligonucleotide molecules comprise nucleotidesequences selected from the group consisting of SEQ ID NOS: 2 to 47, orthe complements of said sequences.

4. BRIEF DESCRIPTION OF THE DRAWINGS

[0041]FIG. 1. Genomic organization of the feline immunodeficiency virus.

[0042]FIG. 2. Expression vector pCMV-MCS.

[0043]FIG. 3. Expression vector pCMV-HA.

[0044]FIG. 4. Plasma viral load detected by virus isolation in thevarious vaccine treatment groups.

[0045]FIG. 5. Plasma viral load detected by QcRT-PCR in the variousvaccine treatment groups.

[0046]FIG. 6. Number of time points at which virus titer was above 10TCID₁₀₀ in the various vaccine treatment groups, as detected by virusisolation.

[0047]FIG. 7. Number of time points at which virus titer was above 10TCID₁₀₀ in the various vaccine treatment groups, as detected byQcRT-PCR.

5. DETAILED DESCRIPTION OF THE INVENTION 5.1. DNA Vaccin 5.1.1.Polynucleotid M I Cules

[0048] As used herein, the terms “DNA”, “RNA”, “gene,” “polynucleotidemolecule,” “nucleotide sequence,” “coding sequence,” and “coding region”are intended to include both DNA and RNA polynucleotide molecules, andto refer to both single-stranded and double-stranded polynucleotidemolecules. Thus, the term “DNA vaccine”, as used herein, encompassesvaccines comprising either DNA or RNA, or both. Also, as used herein,the terms “gene,” “coding sequence,” and “coding region” are intended torefer to polynucleotide molecules that can be transcribed and translated(DNA), or translated (RNA), into an FIV structural or non-structuralprotein in a cat or in an appropriate in vitro host cell expressionsystem when placed in operative association with appropriate regulatoryelements. Polynucleotide molecules of the vaccine composition caninclude, but are not limited to, one or more prokaryotic sequences,eukaryotic sequences, cDNA sequences, genomic DNA sequences (exonsand/or introns), and chemically synthesized DNA and RNA sequences, orany combination thereof.

[0049] The genome of FIV consists of RNA that is reverse transcribedinto DNA and integrated into the genome of an infected feline host.Unless otherwise indicated, all references made herein to specific FIVgenes and nucleotide sequences and, more generally, to polynucleotidemolecules and nucleotide sequences, are intended to encompass both RNAsequences and DNA sequences that correspond thereto according to thecomplementary relationship between RNA and DNA sequences, as well as thecomplements of all such sequences.

[0050] Design, production and manipulation of the polynucleotidemolecules, oligonucleotide molecules and expression vectors disclosedherein are within the skill in the art and can be carried out accordingto known genetic techniques which are described, among other places, inManiatis et al., 1989, Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Ausubel etal., 1989, above; Sambrook et al., 1989, Molecular Cloning: A LaboratoryManual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.; Innis et al. (eds), 1995, PCR Strategies, Academic Press, Inc.,San Diego; and Erlich (ed), 1992, PCR Technology, Oxford UniversityPress, New York, which are incorporated herein by reference.

[0051] The present invention provides a vaccine composition againstfeline immunodeficiency virus (FIV), comprising an immunologicallyeffective amount of a polynucleotide molecule comprising a nucleotidesequence selected from a portion of the genome of an FIV strain, or anucleotide sequence which is a degenerate variant thereof; and aveterinarily acceptable carrier. The FIV strain can be any strain of FIVcurrently known, or any strain to be isolated and identified in thefuture, and is preferably a pathogenic strain, In a preferredembodiment, the FIV strain is FIV-141, which has a genomic RNA sequencecorresponding to the DNA sequence shown in SEQ ID NO:1 (from nt 1 to nt9464). An infectious FIV-141 molecular plasmid clone and the FIV-141virus were deposited on Jul. 1, 1998 with the American Type CultureCollection, and were assigned ATCC Accession Nos. 203001 and VR-2619,respectively. Alternatively, strains of FIV can be isolated from organs,tissues or body fluids of infected cats and propagated in tissue cultureusing standard isolation and tissue culture techniques such as thosedescribed in the publications reviewed above, and any such strains canbe used to isolate polynucleotide molecules necessary to practice thepresent invention.

[0052] Reference is made to FIG. 1, which presents the overall genomicorganization of FIV, and to the genomic sequence of FIV-141 presented inSEQ ID NO:1. In SEQ ID NO:1, the 540 LTR is from nt 1 to nt 354; the GAGpolyprotein gene is from nt 627 to nt 1976; the POL polyprotein gene isfrom nt 1880 to nt 5239; Vif is from nt 5232 to nt 5987; ORF2 is from nt5988 to nt 6224; the ENV gene is from nt 6262 to nt 8826; Rev is from nt6262 to nt 6505, and from nt 8947 to nt 9161; and the 3′ LTR is from nt9111 to nt 9464. Within the GAG polyprotein gene, MA is encoded from nt627 to nt 1031; CA is encoded from nt 1032 to nt 1724; and NC is encodedfrom nt 1725 to nt 1976. Within the POL polyprotein gene, PR is encodedfrom nt 1979 to nt 2326; RT is encoded from nt 2327 to nt 3994; DU isencoded from nt 3995 to nt 4393; and IN is encoded from nt 4394 to nt5239. Within the ENV polyprotein gene, SU is encoded from nt 6262 to nt8088; and TM is encoded from nt 8089 to nt 8826. The nucleotideboundaries presented herein also serve as a guide for selectingcorresponding genes and coding regions of other FIV strains.

[0053] For polynucleotide molecules encoding structural ornon-structural FIV proteins, including, e.g., a GAG protein, POLprotein, ENV protein, ORF2, Vif, or Rev, or substantial portionsthereof, a nucleotide sequence useful in practicing the presentinvention can be any sequence which encodes the particular protein orpolypeptide, i.e., either the native nucleotide sequence found in theparticular FIV genome or, alternatively, a degenerate variant, i.e., anucleotide sequence that encodes the same protein or polypeptide, butwhich differs from the native sequence as based on the degeneracy of thegenetic code. The present invention encompasses vaccine compositions andmethods of using polynucleotide molecules having any of these nucleotidesequences.

[0054] In a preferred embodiment, the polynucleotide molecule of thevaccine composition comprises a nucleotide sequence encoding one or moreof a structural or non-structural protein from an FIV strain, or acombination thereof. The structural protein is selected from the groupconsisting of a GAG protein and an ENV protein. The non-structuralprotein is selected from the group consisting of a POL protein and aregulatory protein. The GAG protein is selected from the groupconsisting of the GAG polyprotein and its substituent proteins, i.e.,MA, CA and NC. The ENV protein is selected from the group consisting ofthe ENV polyprotein and its substituent proteins, i.e., SU and TM. ThePOL protein is selected from the group consisting of the POL polyproteinand its substituent proteins, i.e., PR, RT, DU and IN. The regulatoryprotein is selected from the group consisting of Rev, Vif, ORF2 and LTR.

[0055] In a preferred embodiment, the vaccine composition of the presentinvention comprises a polynucleotide molecule comprising a nucleotidesequence encoding an FIV protein selected from the group consisting ofGAG, MA, CA, NC, ENV, SU, TM, DU and PR.

[0056] In a more preferred embodiment, the vaccine composition of thepresent invention is a combination vaccine, which comprises one or morepolynucleotide molecules having nucleotide sequences encoding acombination of FIV proteins. In a preferred embodiment, the one or morepolynucleotide molecules of the vaccine composition comprise nucleotidesequences encoding at least two different FIV proteins selected from FIVstructural and FIV non-structural proteins, provided that when the oneor more polynucleotide molecules encode the ENV and NC proteins fromFIV, they also encode at least one, more preferably at least two, andmost preferably at least three other FIV structural or non-structuralproteins.

[0057] In a further preferred embodiment, the one or more polynucleotidemolecules of the vaccine composition comprise nucleotide sequencesencoding at least two different GAG proteins from FIV.

[0058] In a further preferred embodiment, the one or more polynucleotidemolecules of the vaccine composition comprise nucleotide sequencesencoding at least one FIV structural protein and at least one FIVnon-structural protein.

[0059] In a further preferred embodiment, the one or more polynucleotidemolecules of the vaccine composition comprise nucleotide sequencesencoding at least three different FIV proteins selected from among theFIV structural and FIV non-structural proteins, i.e., the proteins canbe either all structural proteins or all non-structural proteins, or acombination of structural and non-structural proteins. In a furtherpreferred embodiment, the one or more polynucleotide molecules of thevaccine composition comprise nucleotide sequences encoding at least fourdifferent FIV proteins selected from among the FIV structural and FIVnon-structural proteins. In a further preferred embodiment, the one ormore polynucleotide molecules of the vaccine composition comprisenucleotide sequences encoding at least five different FIV proteinsselected from among the FIV structural and FIV non-structural proteins.In a further preferred embodiment, the one or more polynucleotidemolecules of the vaccine composition comprise nucleotide sequencesencoding at least six different FIV proteins selected from among the FIVstructural and FIV non-structural proteins. In a further preferredembodiment, the one or more polynucleotide molecules of the vaccinecomposition comprise nucleotide sequences encoding at least sevendifferent FIV proteins selected from among the FIV structural and FIVnon-structural proteins.

[0060] In a further preferred embodiment, the one or more polynucleotidemolecules of the vaccine composition comprise a nucleotide sequenceencoding at least one FIV structural protein and a nucleotide sequenceencoding at least one FIV regulatory protein. In a further preferredembodiment, the one or more polynucleotide molecules of the vaccinecomposition comprise a nucleotide sequence encoding at least one FIV POLprotein and a nucleotide sequence encoding at least one FIV regulatorygene. In a further preferred embodiment, the one or more polynucleotidemolecules of the vaccine composition comprise a nucleotide sequenceencoding at least one FIV structural protein, a nucleotide sequenceencoding at least one FIV POL protein, and a nucleotide sequenceencoding at least one FIV regulatory protein.

[0061] In a further preferred embodiment, when the one or morepolynucleotide molecules of the vaccine composition comprise nucleotidesequences encoding a GAG protein, PR protein or ENV protein from FIV, ora combination thereof, nucleotide sequences encoding at least one,preferably at least two, and most preferably at least three, other FIVproteins are present.

[0062] In a particularly preferred embodiment, the vaccine compositionof the present invention comprises one or more polynucleotide moleculescomprising nucleotide sequences encoding a combination of FIV proteins,which combination is selected from the group consisting of GAG/MA/CA/NC;GAG/ENV; GAG/MA/CA/NC/ENV/SU/TM; MA/CA/NC; GAG/MA/NC/DU/PR; andMA/CA/NC/SU/TM. When the vaccine composition of the present invention isa combination vaccine, the nucleotide sequences encoding the various FIVproteins or polypeptides can be on the same polynucleotide molecule, ondifferent polynucleotide molecules, or a combination thereof.

[0063] The vaccine composition may alternatively or additionallycomprise one or more polynucleotide molecules comprising a nucleotidesequence which is a substantial portion of any of the aforementionednucleotide sequences. As used herein, a nucleotide sequence is a“substantial portion” of a nucleotide sequence encoding a GAG protein,POL protein , ENV protein or regulatory protein from an FIV strain,where the nucleotide sequence consists of less than the completenucleotide sequence encoding the particular full length FIV protein, butis at least about 30%, more preferably at least about 50%, and mostpreferably at least about 70% of the complete nucleotide sequenceencoding the particular full length FIV protein, or a degenerate variantthereof, and is useful in practicing the present invention. In apreferred embodiment, the “substantial portion” of the nucleotidesequence encodes at least one epitope of an FIV antigen.

[0064] As used herein, a polynucleotide molecule is “useful inpracticing the present invention” where: (1) the polynucleotidemolecule, upon administration to a cat, can detectably induce aprotective immune response against FIV, or can detectably enhance theinduction of a protective immune response against FIV whenco-administered to a cat with one or more other FIV antigen-encodingpolynucleotide molecules or FIV proteins or polypeptides; (2) thepolynucleotide molecule can be utilized in a recombinant in vitroexpression system to prepare a protein or polypeptide which, uponadministration to a cat can detectably induce a protective immuneresponse against FIV, or can detectably enhance the induction of aprotective immune response against FIV when co-administered to a catwith one or more FIV antigen-encoding polynucleotide molecules or one ormore other FIV proteins or polypeptides; (3) the polynucleotidemolecule, or the protein or polypeptide which is encoded thereby, can beused to induce the production of anti-FIV antibodies in a mammal; or (4)the polynucleotide molecule or its complement can be used as adiagnostic reagent to detect the presence of an FIV-specificpolynucleotide molecule in a fluid or tissue sample from an FIV-infectedcat. Such polynucleotide molecules can be prepared and identified usingstandard techniques known in the art.

[0065] As used herein, a protein or polypeptide is “useful in practicingthe present invention” where: (1) the protein or polypeptide, uponadministration to a cat, can detectably induce a protective immuneresponse against FIV, or can detectably enhance the induction of aprotective immune response against FIV when co-administered to a catwith one or more FIV antigen-encoding polynucleotide molecules or one ormore other FIV proteins or polypeptides; (2) the polypeptide can be usedto induce the production of anti-FIV antibodies in a mammal; or (3) thepolypeptide can be used as a diagnostic reagent to detect the presenceof FIV-specific antibodies in a fluid or tissue sample from anFIV-infected cat. Such polypeptides can be prepared and identified usingstandard techniques known in the art.

[0066] The polynucleotide molecule of the vaccine composition mayalternatively or additionally comprise a nucleotide sequence encoding apolypeptide otherwise having the amino acid sequence of one or more of aGAG protein, POL protein, ENV protein or regulatory protein from an FIVstrain, but in which one or more amino acid residues present in thenative FIV protein has been conservatively substituted with a differentamino acid residue, where the polynucleotide molecule is useful inpracticing the present invention, as usefulness is defined above.Conservative amino acid substitutions, the nucleotide sequences thatencode them, and the methods to prepare them are well known in the art.For example, a polynucleotide molecule can be prepared which encodes theconservative substitution of one or more amino acid residues of anFIV-141 protein, where the resulting polynucleotide molecule or encodedpolypeptide is useful in practicing the present invention. Rules formaking such substitutions include those described by Dayhof, M. D.,1978, Nat. Biomed. Res. Found., Washington, D.C., Vol. 5, Sup. 3, amongothers. More specifically, conservative amino acid substitutions arethose that generally take place within a family of amino acids that arerelated in their side chains. Genetically encoded amino acids aregenerally divided into four groups: (1) acidic=aspartate, glutamate; (2)basic=lysine, arginine, histidine; (3) non-polar=alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and(4) uncharged polar=glycine, asparagine, glutamine, cysteine, serine,threonine, tyrosine. Phenylalanine, tryptophan and tyrosine are alsojointly classified as aromatic amino acids. One or more replacementswithin any particular group, e.g., of a leucine by isoleucine or valine,or of an aspartate by glutamate, or of a threonine by serine, or of anyother amino acid residue by a structurally related amino acid residue,will generally have an insignificant effect on the usefulness of theresulting polypeptide in practicing the present invention. In apreferred embodiment, such a polypeptide having one or more conservativeamino acid substitutions, as encoded by the polynucleotide molecule ofthe present invention, has at least about 70%, more preferably at leastabout 80% and most preferably at least about 90% sequence identity tothe corresponding native FIV protein or polypeptide, and is useful inpracticing the present invention.

[0067] In an alternative embodiment, the one or more polynucleotidemolecules of the vaccine composition comprise a combination of any ofthe aforementioned nucleotide sequences.

[0068] The vaccine composition of the present invention can comprise atleast two different polynucleotide molecules, wherein the firstpolynucleotide molecule comprises a nucleotide sequence encoding one ormore FIV proteins or polypeptides as described above; and the secondpolynucleotide molecule comprises a nucleotide sequence encoding one ormore FIV proteins or polypeptides that are different from those encodedby the first polynucleotide molecule, or encoding a different antigenuseful in detectably inducing a protective response in cats eitheragainst FIV or against a different feline pathogen such as, e.g., felineleukemia virus (FeLV), feline calicivirus, or feline herpes virus, asknown in the art.

[0069] Any of the polynucleotide molecules of the present invention canfurther comprise a nucleotide sequence encoding an immunomodulatorymolecule such as a cytokine or a carrier protein, or the nucleotidesequence encoding the immunomodulatory molecule can be present on adifferent polynucleotide molecule which can be co-administered with apolynucleotide molecule of the present invention.

[0070] The polynucleotide molecule of the vaccine composition can eitherbe DNA or RNA, although DNA is preferred, and is preferably administeredto a cat in an expression vector construct, such as a recombinantplasmid or viral vector, as known in the art. Examples of recombinantviral vectors include recombinant adenovirus vectors and recombinantretrovirus vectors. However, a preferred vaccine formulation comprises anon-viral DNA vector, most preferably a DNA plasmid-based vector. Thepolynucleotide molecule may be associated with lipids to form, e.g.,DNA-lipid complexes, such as liposomes or cochleates. See, e.g.,International Patent Publications WO 93/24640 and WO 98/58630, which areincorporated herein by reference.

[0071] An expression vector useful as a vaccinal agent in a DNA vaccinewill preferably comprise any of the aforementioned polynucleotidemolecules of the present invention having an FIV-related nucleotidesequence. In a preferred embodiment, the expression vector comprises atleast a nucleotide sequence encoding one or more antigenic FIV proteins,or a substantial portion of such a nucleotide sequence, in operativeassociation with one or more transcriptional regulatory elementsrequired for expression of the FIV coding sequence in a eukaryotic cell,such as, e.g., a promoter sequence, as known in the art. In a preferredembodiment, the regulatory element is a strong viral promoter such as,e.g., a viral promoter from RSV, CMV, or SV40, or the LTR promoter froma retrovirus, as known in the art. Such an expression vector alsopreferably includes a bacterial origin of replication and a prokaryoticselectable marker gene for cloning purposes, and a polyadenylationsequence to ensure appropriate termination of the expressed mRNA. Asignal sequence may also be included to direct cellular secretion of theexpressed protein.

[0072] The requirements for expression vectors useful as vaccinal agentsin DNA vaccines are further described, among other places, in U.S. Pat.Nos. 5,703,055, 5,580,859, 5,589,466, International Patent PublicationWO 98/35562, and in various scientific publications, including Ramsay etal., 1997, Immunol. Cell Biol. 75:360-363; Davis, 1997, Cur. OpinionBiotech. 8:635-640; Manickan et al., 1997, Critical Rev. Immunol.17:139-154; Robinson, 1997, Vaccine 15(8):785-787; Robinson et al.,1996, AIDS Res. Hum. Retr. 12(5):455-457; Lai and Bennett, 1998,Critical Rev. Immunol. 18:449-484; and Vogel and Sarver, 1995, Clin.Microbiol. Rev. 8(3):406-410, which are incorporated herein byreference.

5.1.2. DNA Vaccine Formulation and Use

[0073] The present invention further provides a method of preparing avaccine composition against FIV, comprising combining an immunologicallyeffective amount of any one or more of the aforementioned FIV-relatedpolynucleotide molecules, or any one or more expression vectorscomprising such polynucleotide molecules, with a veterinarily acceptablecarrier in a form suitable for administration to a cat.

[0074] As used herein, the term “immunologically effective amount”, asit relates to an FIV-related polynucleotide molecule, expression vector,protein or polypeptide, refers to that amount of polynucleotidemolecule, expression vector, protein or polypeptide, respectively,capable of inducing, or enhancing the induction of, a protectiveresponse against FIV when administered to a cat.

[0075] As used herein, the phrase “capable of inducing a protectiveresponse against FIV”, and the like, is used broadly to include theinduction of any immune-based response in the cat in response tovaccination, including either an antibody or cell-mediated immuneresponse, or both, that serves to protect the vaccinated animal againstFIV.

[0076] As used herein, a polynucleotide molecule, expression vector,protein or polypeptide “can enhance the induction of a protective immuneresponse against FIV” when its addition to an FIV vaccine compositioncomprising one or more other FIV antigen-encoding polynucleotidemolecules, proteins or polypeptides serves to detectably increase theprotective response against FIV that would otherwise be induced by avaccine combination without such an addition.

[0077] The terms “protective response” and “protect” as used hereinrefer not only to the absolut prevention of any of the symptoms orconditions resulting from FIV infection in cats, but also to anydetectable delay in the onset of any such symptoms or conditions, anydetectable reduction in the degree or rate of infection by FIV, or anydetectable reduction in the severity of the disease or any symptom orcondition resulting from infection by FIV, including, e.g., anydetectable reduction in viral load, CD4/CD8 T lymphocyte ratio,mortality, etc., as compared to an FIV-infected animal (i.e., acontrol). The immunologically effective amount of the one or morepolynucleotide molecules, expression vectors, or FIV proteins orpolypeptides, may be administered either in a single dose or in divideddoses. For purposes of the present invention, a protective response isdeemed to have been induced if it can be detected after administrationof the single complete dose, or after administration to the animal ofall of the divided doses. The present invention further encompasses theadministration to a previously vaccinated cat of an additional “booster”dose to increase the protective response against FIV.

[0078] In a preferred embodiment, the vaccine composition of the presentinvention is capable of inducing a protective response in a cat againsthomologous challenge, i.e., a cat vaccinated with the vaccinecomposition exhibits a protective response against a strain of the samesub-type of FIV from which the antigenic components of the vaccinecomposition were prepared or derived.

[0079] In a more preferred embodiment, the vaccine composition of thepresent invention is capable of inducing a protective response in a catagainst heterologous challenge, i.e., a cat vaccinated with the vaccinecomposition exhibits a protective response against a strain of adifferent sub-type of FIV from which the antigenic components of thevaccine composition were prepared or derived.

[0080] The vaccine composition of the present invention will typicallybe adapted for intradermal or intramuscular injection, although otherroutes (e.g., intravenous, intraperitoneal, intranasal, oral,intraocular, rectal, vaginal) can also be effective. Veterinarilyacceptable carriers can be any carriers known in the art that arecompatible with DNA vaccines, as described in the publications citedherein. The vaccine composition of the present invention can beformulated following accepted convention using standard buffers,carriers, stabilizers, diluents, preservatives and/or solubilizers, andcan also be formulated to facilitate sustained release. For example, thepolynucleotide molecule of the vaccine composition can be prepared inaqueous solution, such as in sterile saline or PBS solution, orincorporated into liposomes or cochleates for parenteral administration.See, e.g., International Patent Publication WO 93/24640 or U.S. Pat. No.5,703,055. Suitable other vaccine vehicles and additives that areparticularly useful in DNA vaccine formulations are known or will beapparent to those of skill in the art. Alternatively, the polynucleotidemolecule of the vaccine composition can be coated onto metallicparticles, such as gold particles, for administration to a cat using a“gene gun.” See, e.g., Tang et al., 1992, Nature 356:152-154. Thus, forpurposes of this invention, metallic particles, such as gold particles,onto which polynucleotide molecules or expression vectors of the presentinvention can be coated and administered to cats are also considered tobe a veterinarily acceptable carrier. Alternatively, the polynucleotidemolecule of the vaccine composition can be prepared for oraladministration and targeted to the Peyer's patches, such as bymicroencapsulation, e.g., with poly(lactide-co-glycolide) (PLG),preferably into microparticles of ≦10 um in diameter, as known in theart. See, e.g., Jones et al., 1998, in: Brown and Haaheim (eds.):Modulation of the Immune Response to Vaccine Antigens, Dev Biol Stand.Basel, Karger, 92:149-155.

[0081] The vaccine composition of the present invention can furthercomprise a supplemental component selected from the group consisting ofan adjuvant, a cytokine, a polynucleotide molecule comprising anucleotide sequence encoding an immunomodulatory molecule such as acytokine or carrier protein which can enhance or modulate the immuneresponse against FIV, an agent that facilitates uptake by feline cellsof the polynucleotide molecule, such as, e.g., bupivacaine, or acombination thereof. The nucleotide sequence encoding theimmunomodulatory molecule can either be situated on the samepolynucleotide molecule or expression vector as the nucleotide sequenceof the FIV antigen or on a separate polynucleotide molecule orexpression vector which is preferably co-administered with, oradministered at about the same time as, the polynucleotide moleculecomprising the nucleotide sequence of the FIV antigen. The use of DNAvaccines in combination with cytokines, including, e.g., interleukinsand interferons, is described in Lee et al., 1999, Vaccine 17:473-479;Okada et al., 1997, J. Immunol. 159:3638-3647; Sin et al., 1997, Vaccine15:1827-1833; Chow et al., 1997, J. Virol. 71:169-178; Tsuji et al.,1997, J. Immunol. 158:158:4008-4013; and Kim et al., 1997, J. Immunol.158:816-826, among others, which are incorporated herein by reference.

[0082] Adjuvants that can be used in the vaccine of the presentinvention are those which are compatible with DNA vaccines as known inthe art. A non-limiting example of an adjuvant designed for DNA vaccinescomprises a negatively charged, mineral-based particle preparation, asdescribed in International Patent Publication WO 98/35562.

[0083] The polynucleotide molecules or expression vectors of the vaccinecomposition can be stored frozen and thawed prior to administration or,more preferably, in lyophilized form and rehydrated prior toadministration using a sterile diluent as known in the art.

[0084] Polynucleotide molecules and expression vectors of the vaccinecomposition of the present invention can be microencapsulated to improveadministration and efficacy. For example, methods for encapsulating DNAfor oral delivery are described in Jones et al., 1998, above.

[0085] The present invention further provides a method of vaccinating acat against FIV, comprising administering to the cat a vaccinecomposition of the pres nt invention. The vaccine is preferablyadministered parenterally, e.g., either by subcutaneous, intramuscularor intradermal injection. However, the vaccine may instead beadministered by intraperitoneal or intravenous injection, or by otherroutes, including, e.g., orally, intranasally, rectally, vaginally,intra-ocularly, or by a combination of routes, and also by delayedrelease devices as known in the art. The skilled artisan will be able toformulate the vaccine composition according to the route chosen.

[0086] An effective dosage of the polynucleotide molecule or expressionvector of the present invention can be determined by conventional means,starting with a low dose of the polynucleotide molecule or expressionvector, and then increasing the dosage while monitoring the effects.Numerous factors can be taken into consideration when determining anoptimal dose per cat. Primary among these is the size, age and generalcondition of the cat, the presence of other drugs in the cat, thevirulence of a particular strain of FIV against which the cat is beingvaccinated, and the like. The actual dosage is preferably chosen afterconsideration of the results from other animal studies.

[0087] Vaccine regimens can be selected based on the above-describedfactors. The vaccine of the invention can be administered at any timeduring the life of a particular cat depending upon several factorsincluding, e.g., the timing of an outbreak of FIV among other cats. Thevaccine can be administered to cats of weaning age or younger, or tomore mature animals. Effective protection may require only a primaryvaccination, or one or more booster vaccinations may also be needed. Adose that provides adequate protection can be determined empirically bychallenging vaccinated and unvaccinated cats (control) with FIV andmonitoring and comparing disease progression, including any indicatorthereof as known in the art, in the two groups of animals. The timing ofvaccination and the number of boosters, if any, will preferably bedetermined by a veterinarian based on analysis of all relevant factors,some of which are described above.

[0088] The concentration of the polynucleotide molecule or expressionvector of the present invention in the vaccine preferably ranges fromabout 0.05 μg/ml to about 10 mg/ml, and more preferably from about 0.5μg/ml to about 5.0 mg/ml. A suitable dosage volume ranges from about 0.1and 5 ml, which may be administered in a single dose, or in divideddoses.

[0089] The present invention further provides a kit for vaccinating acat against FIV, comprising a first container comprising animmunologically effective amount of any one or more of theaforementioned polynucleotide molecules or expression vectors of thepresent invention. In a non-limiting embodiment, the polynucleotidemolecule or expression vector is stored in the first container inlyophilized form. The kit may optionally further comprise a secondcontainer comprising a sterile diluent which can be used to dilute orrehydrate the polynucleotide molecule or expression vector in the firstcontainer.

5.2. Antibodies

[0090] The present invention further provides an isolated antibody thatbinds specifically to an FIV protein. In a preferred embodiment, theantibody is produced in a mammal in response to administration of apolynucleotide molecule or expression vector having a nucleotidesequence encoding the FIV protein or an epitope thereof, such as, e.g.,a polynucleotide molecule or expression vector as present in the vaccinecomposition of the present invention.

[0091] Antibodies can be raised in a host animal in response toadministration of the vaccine composition of the present invention oragainst an expressed or purified FIV antigen, and isolated using knownmethods. Various host animals, including cats, dogs, pigs, cows, horses,rabbits, goats, sheep, and mice, can be immunized with the vaccinecomposition of the present invention or with a partially orsubstantially purified FIV antigen. An adjuvant, such as those listedbelow in Section 5.3.4, can be selected and used to enhance antibodyproduction. Polyclonal antibodies can be obtained from the serum of theimmunized animal, tested for anti-FIV protein specificity, and isolatedusing standard techniques. Alternatively, monoclonal antibodies againstthe FIV protein can be prepared and isolated using any technique thatprovides for the production of antibody molecules by continuous celllines in culture. These include but are not limited to the hybridomatechnique originally described by Kohler and Milstein (Nature, 1975,256: 495-497); the human B-cell hybridoma technique (Kosbor et al.,1983, Immunology Today 4:72; Cote et al., 1983, Proc. Natl. Acad. Sci.USA 80: 2026-2030); and the EBV-hybridoma technique (Cole et al., 1985,Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Alternatively, techniques described for the production of singlechain antibodies (see, e.g., U.S. Pat. No. 4,946,778) can be adapted toproduce FIV antigen-specific single chain antibodies. These publicationsare incorporated herein by reference.

[0092] Antibody fragments that contain specific binding sites for an FIVantigen are also encompassed within the present invention, and can begenerated and isolated by known techniques. Such fragments include butare not limited to F(ab′)₂ fragments which can be generated by pepsindigestion of an intact antibody molecule, and Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries can be constructed (Huse et al.,1989, Science 246: 1275-1281) to allow rapid identification of Fabfragments having the desired specificity to the FIV antigen.

[0093] Techniques for the production and isolation of monoclonalantibodies and antibody fragments are well-known in the art, and areadditionally described, among other places, in Harlow and Lane, 1988,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, and inJ. W. Goding, 1986, Monoclonal Antibodies: Principles and Practice,Academic Press, London, which are incorporated herein by reference.

5.3. FIV Protein-Based Vaccines

[0094] The present invention further provides a vaccine compositionagainst FIV, comprising an immunologically effective amount of a GAGprotein, POL protein, ENV protein, regulatory protein, or a combinationthereof, from an FIV strain. The FIV strain can be any strain of FIV,but is preferably strain FIV-141. The GAG protein is selected from thegroup consisting of the GAG polyprotein and its substituent proteins,i.e., MA, CA and NC. The POL protein is selected from the groupconsisting of the POL polyprotein and its substituent proteins, i.e.,PR, RT, DU and IN. The ENV protein is selected from the group consistingof the ENV polyprotein and its substituent proteins, i.e., SU and TM.The regulatory protein is selected from the group consisting of Rev, Vifand ORF2.

[0095] In a more preferred embodiment, the vaccine composition of thepresent invention comprises a combination of FIV proteins. In apreferred embodiment, the proteins of the vaccine composition compriseat least two different FIV proteins selected from among the FIVstructural and FIV non-structural proteins. In a further preferredembodiment, the proteins of the vaccine composition comprise at leasttwo different GAG proteins from FIV. In a further preferred embodiment,the proteins of the vaccine composition comprise at least one FIVstructural protein and at least one FIV non-structural protein. In afurther preferred embodiment, the proteins of the vaccine compositioncomprise at least three different FIV proteins selected from among theFIV structural and FIV non-structural proteins. In a further preferredembodiment, the proteins of the vaccine composition comprise at leastfour different FIV proteins selected from among the FIV structural andFIV non-structural proteins. In a further preferred embodiment, theproteins of the vaccine composition comprise at least five different FIVproteins selected from among the FIV structural and FIV non-structuralproteins. In a further preferred embodiment, the proteins of the vaccinecomposition comprise at least six different FIV proteins selected fromamong the FIV structural and FIV non-structural proteins. In a furtherpreferred embodiment, the proteins of the vaccine composition compriseat least seven different FIV proteins selected from among the FIVstructural and FIV non-structural proteins.

[0096] In a particularly preferred embodiment, the combination of FIVproteins is selected from the group consisting of GAG/MA/CA/NC; GAG/ENV;GAG/MA/CA/NC/ENV/SU/TM; MA/CA/NC; GAG/MA/NC/DU/PR; and MA/CA/NC/SU/TM.

[0097] The vaccine composition can alternatively comprise: (a) at leastone polypeptide which is homologous to any of the aforementioned FIVproteins; (b) at least one peptide fragment of any of the aforementionedFIV proteins, or homologous polypeptides of (a); (c) at least one fusionprotein comprising any of the aforementioned FIV proteins, homologouspolypeptides of (a) or peptide fragments of (b) fused to a fusionpartner; (d) at least one of an analog or derivative of any of theaforementioned FIV proteins, homologous polypeptides of (a), peptidefragments of (b), or fusion proteins of (c); or (e) a combinationthereof.

[0098] As used herein, the term “homologous” refers to a polypeptideotherwise having the amino acid sequence of an FIV protein orpolypeptide, but in which one or more amino acid residues have beenconservatively substituted with a different amino acid residue asdefined above in Section 5.1.1, where the resulting polypeptide isuseful in practicing the present invention as usefulness is definedtherein. In a preferred embodiment, such a polypeptide has at leastabout 70%, more preferably at least about 80%, and most preferably atleast about 90% sequence identity to a native FIV protein orpolypeptide.

[0099] As used herein, a “peptide fragment” of an FIV protein refers toa polypeptide consisting of less than the complete amino acid sequenceof the corresponding full-length FIV protein, but comprising asub-sequence of at least about 10 amino acid residues, more preferablyat least about 20 amino acid residues, and most preferably at leastabout 30 amino acid residues of the amino acid sequence thereof, andthat is useful in practicing the present invention as defined above. Ina preferred embodiment, a peptide fragment of an FIV protein comprisesthe amino acid sequence of an epitope of the FIV protein against whichantibodies can be raised.

[0100] As used herein, a “fusion protein” comprises an FIV protein,homologous polypeptide or peptide fragment of the present inventionjoined to a carrier or fusion partner, which fusion protein is useful inpracticing the present invention, as usefulness is defined above forpolypeptides. See Section 5.3.1 below for examples of fusion partners.Fusion proteins are useful for a variety of reasons, including toincrease the stability of recombinantly-expressed FIV polypeptides, asdistinct antigenic components in an FIV vaccine, to enhance theinduction of antisera against the particular FIV antigen partner, tostudy the biochemical properties of the FIV antigen partner, to serve asdiagnostic reagents, or to aid in th identification or purification ofthe expressed FIV antigen partner as described below.

[0101] Fusion proteins of the present invention can be engineered usingstandard techniques to further contain specific protease cleavage sitesso that the particular FIV antigen partner can be released from thecarrier or fusion partner by treatment with a specific protease. Forexample, a fusion protein of the present invention can further comprisea thrombin or factor Xa cleavage site, among others.

[0102] The present invention further provides analogs and derivatives ofan FIV protein, homologous polypeptide, peptide fragment or fusionprotein, where such analogs and derivatives are useful in practicing thepresent invention, as usefulness is defined above for polypeptides.Manipulations that result in the production of analogs can be carriedout either at the gene level or at the protein level, or both, toimprove or otherwise alter the biological or immunologicalcharacteristics of the particular polypeptide from which the analog isprepared. For example, at the gene level, a cloned DNA molecule encodingan FIV protein can be modified by one or more known strategies to encodean analog of that protein. Such modifications include, but are notlimited to, endonuclease digestion, and mutations that create or destroytranslation, initiation or termination sequences, or that createvariations in the coding region, or a combination thereof. Suchtechniques are described, among other places, in Maniatis et al., 1989,above; Ausubel et al., 1989, above; Sambrook et al., 1989, above; Inniset al (eds), 1995, above; and Erlich (ed), 1992, above.

[0103] Alternatively or additionally, an analog of the present inventioncan be prepared by modification of an FIV protein or other polypeptideof the present invention at the protein level. Chemical modifications ofthe protein can be carried out using known techniques, including but notlimited to one or more of the following: substitution of one or moreL-amino acids of the protein with corresponding D-amino acids, aminoacid analogs, or amino acid mimics, so as to produce, e.g., carbazatesor tertiary centers; or specific chemical modification, such asproteolytic cleavage with, e.g., trypsin, chymotrypsin, papain or V8protease, or treatment with NaBH₄ or cyanogen bromide, or acetylation,formylation, oxidation or reduction, etc.

[0104] An FIV protein or other polypeptide of the present invention canbe derivatized by conjugation thereto of one or more chemical groups,including but not limited to acetyl groups, sulfur bridging groups,glycosyl groups, lipids, and phosphates, and/or a second FIV protein orother polypeptide of the present invention, or another protein, such as,e.g., serum albumin, keyhole limpet hemocyanin, or commerciallyactivated BSA, or a polyamino acid (e.g., polylysine), or apolysaccharide, (e.g., sepharose, agarose, or modified or unmodifiedcelluloses), among others. Such conjugation is preferably by covalentlinkage at amino acid side chains and/or at the N-terminus or C-terminusof the FIV protein. Methods for carrying out such conjugation reactionsare well known in the field of protein chemistry.

[0105] Derivatives useful in practicing the claimed invention alsoinclude those in which a water-soluble polymer, such as, e.g.,polyethylene glycol, is conjugated to an FIV protein or otherpolypeptide of the present invention, or to an analog thereof, therebyproviding additional desirable properties while retaining, at least inpart, or improving the immunogenicity of the FIV protein. Theseadditional desirable properties include, e.g., increased solubility inaqueous solutions, increased stability in storage, increased resistancto proteolytic degradation, and increased in vivo half-life.Water-soluble polymers suitable for conjugation to an FIV protein orother polypeptide of the present invention include but are not limitedto polyethylene glycol homopolymers, polypropylene glycol homopolymers,copolymers of ethylene glycol with propylene glycol, wherein saidhomopolymers and copolymers are unsubstituted or substituted at one endwith an alkyl group, polyoxyethylated polyols, polyvinyl alcohol,polysaccharides, polyvinyl ethyl ethers, and α,βpoly[2-hydroxyethyl]-DL-aspartamide. Polyethylene glycol isparticularly preferred. Methods for making water-soluble polymerconjugates of polypeptides are known in the art and are described in,among other places, U.S. Pat. Nos. 3,788,948; 3,960,830; 4,002,531;4,055,635; 4,179,337; 4,261,973; 4,412,989; 4,414,147; 4,415,665;4,609,546; 4,732,863; 4,745,180; European Patent (EP) 152,847; EP98,110; and Japanese Patent (JP) 5,792,435, which patents areincorporated herein by reference.

5.3.1. Recombinant Vectors

[0106] Protein-based FIV vaccines of the present invention can beprepared by recombinant expression of a polynucleotide molecule having anucleotide sequences encoding a particular FIV protein or polypeptide.To carry out such expression, the present invention provides recombinantcloning and expression vectors comprising the polynucleotide molecule.Expression vectors of the present invention are preferably constructedso that the polynucleotide molecule is in operative association with oneor more regulatory elements necessary for transcription and translation.Such expression vectors are useful in an expression system, such as atransformed host cell, to produce a recombinantly-expressed FIV protein.More preferably, expression vectors of the present invention areconstructed so that the polynucleotide molecule is in operativeassociation with one or more regulatory elements necessary fortranscription and translation in a bacterial cell, or in a mammalianhost cell such as a feline cell. In addition to serving as a reagent forthe production of an FIV protein in vitro, an expression vector capableof expression in a feline cell is also useful as a vaccinal agent in aDNA vaccine composition for administration to cats as described above.

[0107] As used herein, the term “regulatory element” includes but is notlimited to nucleotide sequences that encode inducible and non-induciblepromoters, enhancers, operators, and other elements known in the artthat can serve to drive and/or regulate expression of the codingsequence of the polynucleotide molecule. As used herein, thepolynucleotide molecule is in “operative association” with one or moreregulatory elements where the regulatory elements effectively regulateand provide for the transcription of the coding sequence of thepolynucleotide molecule, or the translation of its mRNA, or both.

[0108] A variety of expression vectors are known in the art that can beutilized to express the coding sequence of a polynucleotide molecule ofthe present invention, including recombinant bacteriophage DNA, plasmidDNA and cosmid DNA expression vectors containing the polynucleotidemolecule, for transformation of bacteria or yeast; and recombinant virusexpression vectors such as, e.g., baculovirus containing thepolynucleotide molecule for transfection of insect cells, or adenovirusor vaccinia virus containing the polynucleotide molecule fortransfection of mammalian cells, among others.

[0109] Typical prokaryotic expression vector plasmids that can beengineered to contain a polynucleotide molecule of the present inventioninclude pUC8, pUC9, pBR322 and pBR329 (Biorad Laboratories, Richmond,Calif.), and pPL and pKK223 (Pharmacia, Piscataway, N.J.), among manyothers.

[0110] Typical eukaryotic expression vectors that can be engineered tocontain a polynucleotide molecule according to the present inventioninclude an ecdysone-inducible mammalian expression system (Invitrogen,Carlsbad, Calif.), cytomegalovirus promoter-enhancer-based systems(Promega, Madison, Wis.; Stratagene, La Jolla, Calif.; Invitrogen), andbaculovirus-based expression systems (Promega), among others.

[0111] The regulatory elements of these and other vectors can vary intheir strength and specificities. Depending on the host/vector systemutilized, any of a number of suitable transcription and translationelements can be used. For instance, when cloning in mammalian cellsystems, promoters isolated from the genome of mammalian cells, e.g.,mouse metallothionein promoter, or from viruses that grow in thesecells, e.g., vaccinia virus 7.5K promoter or Moloney murine sarcomavirus long terminal repeat, may be used. Promoters obtained byrecombinant DNA or synthetic techniques may also be used to provide fortranscription of the inserted sequence. In addition, expression fromcertain promoters can be elevated in the presence of particularinducers, e.g., zinc and cadmium ions for metallothionein promoters.

[0112] Non-limiting examples of transcriptional regulatory regions orpromoters include for bacteria, the β-gal promoter, the T7 promoter, theTAC promoter, λ left and right promoters, trp and lac promoters, andtrp-lac fusion promoters; for yeast, glycolytic enzyme promoters, suchas ADH-I and -II promoters, GPK promoter, PGI promoter, and TRPpromoter; for mammalian cells, SV40 early and late promoters, andadenovirus major late promoters.

[0113] Specific initiation signals are also required for sufficienttranslation of inserted FIV coding sequences. These signals typicallyinclude an ATG initiation codon and adjacent sequences. In cases where apolynucleotide molecule, including its own initiation codon and adjacentsequences, is inserted into an appropriate expression vector, noadditional translation control signals may be needed. However, in caseswhere only a portion of a coding sequence is inserted, exogenoustranslational control signals, including an ATG initiation codon, and atranslation stop codon such as TAA, TAG, or TGA, may be required. Theseexogenous translational control signals and initiation codons can beobtained from a variety of sources, both natural and synthetic.Furthermore, the initiation codon must be in phase with the readingframe of the coding region to ensure in-frame translation of the entireinsert.

[0114] The polynucleotide molecule of the expression vector may furthercomprise a nucleotide sequence which encodes an additional polypeptidefused to the FIV antigen. Such an additional polypeptide can be animmunomodulatory molecule such as a cytokine useful to enhance orotherwise modulate the immune response of a cat to which the expressionvector or encoded fusion protein has been administered. The use of DNAvaccines with cytokines, including, e.g., interleukins and interferons,is presented in Lee et al., 1999, above; Okada et al., 1997, above; Sinet al., 1997, above; Chow et al., 1997, above; Tsuji et al., 1997,above; and Kim et al., 1997, above, among others.

[0115] Additional fusion protein expression vectors include vectorsincorporating sequences that encode β-galactosidase and trpE fusions,maltose-binding protein fusions, glutathione-S-transferase fusions andpolyhistidine fusions (carrier regions). Such fusion proteins can beuseful to aid in purification of the expressed protein. For example, anFIV antigen-maltose-binding protein fusion can be purified using amyloseresin; an FIV antigen-glutathione-S-transferase fusion protein can bepurified using glutathione-agarose beads; and an FIVantigen-polyhistidine fusion can be purified using divalent nickelresin. Alternatively, antibodies against a carrier protein or peptidecan be used for affinity chromatography purification of the fusionprotein. For example, a nucleotide sequence coding for the targetepitope of a monoclonal antibody can be engineered into the expressionvector in operative association with the regulatory elements andsituated so that the expressed epitope is fused to the FIV antigen. Forexample, a nucleotide sequence coding for the FLAG™ epitope tag(International Biotechnologies Inc.), which is a hydrophilic markerpeptide, can be inserted by standard techniques into the expressionvector at a point corresponding, e.g., to the amino or carboxyl terminusof the FIV antigen. The expressed FIV antigen-FLAG™ epitope fusionproduct can then be detected and affinity-purified using commerciallyavailable anti-FLAG™ antibodies. In an alternative embodiment, the FIVprotein or other polypeptide is fused to an epitope tag from humaninfluenza hemagglutinin, such as a nine amino acid epitope tag fromhuman influenza hemagglutinin (HA) protein as described below in Section6.4.

[0116] The expression vector can also be engineered to containpolylinker sequences that encode specific protease cleavage sites sothat the expressed FIV antigen can be released from the carrier regionor fusion partner by treatment with a specific protease. For example,the fusion protein vector can include DNA sequences encoding thrombin orfactor Xa cleavage sites, among others.

[0117] A signal sequence upstream from and in reading frame with the FIVantigen coding region can be engineered into the expression vector byknown methods to direct the trafficking and secretion of the expressedprotein. Non-limiting examples of signal sequences include those fromα-factor, immunoglobulins, outer membrane proteins, penicillinase, andT-cell receptors, among others.

[0118] To aid in the selection of host cells transformed or transfectedwith an expression vector of the present invention, the expressionvector can be engineered to further comprise a coding sequence for areporter gene product or other selectable marker. Such a coding sequenceis preferably in operative association with the regulatory elementcoding sequences, as described above. Reporter genes that are useful inthe invention are well known in the art and include those encodingchloramphenicol acetyltransferase (CAT), green fluorescent protein,firefly luciferase, and human growth hormone, among others. Selectablemarkers, and their nucleotide sequences, are well-known in the art, andinclude gene products conferring resistance to antibiotics oranti-metabolites, or that supply an auxotrophic requirement. Examples ofsuch sequences include those that encode thymidine kinase activity, orresistance to erythromycin, ampicillin, or kanamycin, among others.

[0119] Methods are well-known in the art for constructing expressionvectors containing particular coding sequences in operative associationwith appropriate regulatory elements, as well as nucleotide sequencesencoding selectable markers, signal sequences, and fusion partners, andsuch methods may be used to practice the present invention. Such methodsinclude in vitro recombinant techniques, synthetic techniques, and invivo genetic recombination, as described, among other places, inManiatis et al., 1989, above; Ausubel et al., 1989, above; and Sambrooket al., 1989, above.

5.3.2. Transformation of Host Cells

[0120] Expression vectors comprising a polynucleotide molecule ofinterest can be transformed into host cells for propagation, or forexpression of the encoded FIV antigen. Host cells useful in the practiceof the invention can be eukaryotic or prokaryotic. Such transformed hostcells include but are not limited to microorganisms, such as bacteriatransformed with recombinant bacteriophage DNA, plasmid DNA or cosmidDNA expression vectors; or yeast transformed with a recombinantexpression vector; or animal cells, such as insect cells infected with arecombinant virus expression vector, e.g., baculovirus, or mammaliancells, such as feline cells, infected with a recombinant virusexpression vector, e.g., adenovirus or vaccinia virus, among others.

[0121] Bacterial cells for use as host cells include a strain of E. colisuch as, e.g., the DH5α strain, available from the ATCC, Rockville, Md.,USA (Accession No. 31343), or from Stratagene (La Jolla, Calif.).Eukaryotic host cells include yeast cells, although mammalian cells,such as from a cat, mouse, hamster, cow, monkey, or human cell line, mayalso be used effectively. Specific examples of eukaryotic host cellsthat may be used to express the recombinant protein of the inventioninclude Chinese hamster ovary (CHO) cells (e.g., ATCC Accession No.CCL-61), NIH Swiss mouse embryo cells NIH/3T3 (e.g., ATCC Accession No.CRL-1658), Madin-Darby bovine kidney (MDBK) cells (ATCC Accession No.CCL-22), and thymidine kinase-deficient cells, e.g., L-M (T^(−k)) (ATCCAccession No. CCL-1.3) and tk⁻-ts13 (ATCC Accession No. CRL-1632).

[0122] The recombinant expression vector of the invention is preferablytransformed or transfected into one or more host cells of asubstantially homogeneous culture of cells. The expression vector isgenerally introduced into host cells in accordance with knowntechniques, such as, e.g., by calcium phosphate precipitation, calciumchloride treatment, microinjection, electroporation, transfection bycontact with a recombined virus, liposome-mediated transfection,DEAE-dextran transfection, transduction, conjugation, or microprojectilebombardment, among others. Selection of transformants may be conductedby standard procedures, such as by selecting for cells expressing aselectable marker, e.g., antibiotic resistance, associated with therecombinant expression vector.

[0123] Once the expression vector is introduced into the host cell, theintegration and maintenance of the polynucleotide molecule of thepresent invention, either in the host cell genome or episomally, can beconfirmed by standard techniques, e.g., by Southern hybridizationanalysis, restriction enzyme analysis, PCR analysis including reversetranscriptase PCR (RT-PCR), or by immunological assay to detect theexpected protein product. Host cells containing and/or expressing thepolynucleotide molecule of the present invention may be identified byany of at least four general approaches, which are well-known in theart, including: (i) DNA-DNA, DNA-RNA, or RNA-antisense RNAhybridization; (ii) detecting the presence of “marker” gene functions;(iii) assessing the level of transcription as measured by theexpression, e.g., of an FIV-specific mRNA transcript in the host cell;or (iv) detecting the presence of mature polypeptide product, e.g., byimmunoassay, as known in the art.

5.3.3. Expression and Purification of Recombinant Polypeptides

[0124] Once a polynucleotide molecule of interest has been stablyintroduced into an appropriate host cell, the transformed host cell canbe clonally propagated, and the resulting cells grown under conditionsconducive to the maximum production of the encoded FIV antigen. Suchconditions typically include growing transformed cells to high density.Where the expression vector comprises an inducible promoter, appropriateinduction conditions such as, e.g., temperature shift, exhaustion ofnutrients, addition of gratuitous inducers (e.g., analogs ofcarbohydrates, such as isopropyl-β-D-thiogalactopyranoside (IPTG)),accumulation of excess metabolic by-products, or the like, are employedas needed to induce expression.

[0125] Where the expressed FIV antigen is retained inside the hostcells, the cells are harvested and lysed, and the product purified fromthe lysate under extraction conditions known in the art to minimizeprotein degradation such as, e.g., at 4° C., or in the presence ofprotease inhibitors, or both. Where the expressed FIV antigen issecreted from the host cells, the exhausted nutrient medium may simplybe collected and the protein isolated therefrom.

[0126] The expressed FIV antigen can be purified from cell lysates orculture medium, as appropriate, using standard methods, including butnot limited to one or more of the following methods: ammonium sulfateprecipitation, size fractionation, ion exchange chromatography, HPLC,density centrifugation, and affinity chromatography. Where the expressedFIV antigen exhibits enzymatic activity, increasing purity of thepreparation can be monitored at each step of the purification procedureby use of an appropriate enzyme assay, as known in the art. If theexpressed protein lacks biological activity, it may be detected asbased, e.g., on size, or reactivity with an antibody otherwise specificfor the FIV antigen, or by the presence of a fusion tag.

5.3.4. Protein Vaccine Formulation and Use

[0127] The present invention provides a method of preparing a vaccinecomposition against FIV, comprising combining an immunologicallyeffective amount of any of the aforementioned FIV-related proteins orpolypeptides, or a combination thereof, with a veterinarily acceptablecarrier in a form suitable for administration to a cat. Alternatively,any one or more of the aforementioned proteins or polypeptides can becombined with any one or more of the aforementioned polynucleotidemolecules or expression vectors of the present invention, and aveterinarily acceptable carrier, to prepare a combined DNA/proteinvaccine composition.

[0128] As used herein to refer to proteins and polypeptides, the term“immunologically effective amount” refers to that amount of protein orpolypeptide antigen capable of inducing, or enhancing the induction of,a protective response against FIV when administered to a cat aftereither a single administration, or after multiple administrations. Thephrases “capable of inducing a protective response” and “can enhance theinduction of a protective immune response against FIV”, as well as theterms “protective response” and “protect”, and the like, as used hereinare as defined above in Section 5.1.2.

[0129] The vaccine composition of the present invention will typicallybe adapted for parenteral administration, e.g., by intradermal orintramuscular injection, although other routes (e.g., intravenous,intranasal, oral, etc.) can also be effective. Vaccine compositions ofthe present invention can be formulated following accepted conventionusing standard buffers, carriers, stabilizers, diluents, preservatives,and solubilizers, and can also be formulated to facilitate sustainedrelease. Diluents can include water, saline, dextrose, ethanol,glycerol, and the like. Additives for isotonicity can include sodiumchloride, dextrose, mannitol, sorbitol, and lactose, among others.Stabilizers include albumin, among others.

[0130] Adjuvants can optionally be employed in the vaccine. Non-limitingexamples of adjuvants include the RIBI adjuvant system (Ribi Inc.),alum, aluminum hydroxide gel, oil-in-water emulsions, water-in-oilemulsions such as, e.g., Freund's complete and incomplete adjuvants,Block co polymer (CytRx, Atlanta Ga.), SAF-M (Chiron, EmeryvilleCalif.), AMPHIGEN® adjuvant, saponin, Quil A, QS-21 (Cambridge BiotechInc., Cambridge Mass.), or other saponin fractions, SEAM-1,monophosphoryl lipid A, Avridine lipid-amine adjuvant, heat-labileenterotoxin from E. coli (recombinant or otherwise), cholera toxin, ormuramyl dipeptide, among many others. The vaccine can further compriseone or more other immunomodulatory agents such as, e.g., interleukins,interferons, or other cytokines, or a polynucleotide molecule having anucleotide sequence encoding the same.

[0131] Suitable veterinarily acceptable vaccine vehicles, carriers, andadditives are known, or will be apparent to those skilled in the art;see, e.g., Remington's Pharmaceutical Science, 18th Ed., 1990, MackPublishing, which is incorporated herein by reference. The vaccine canbe stored in solution, or alternatively in lyophilized form to bereconstituted with a sterile diluent solution prior to administration.

[0132] The present invention further provides vaccine formulations forthe sustained release of the antigen. Examples of such sustained releaseformulations include the proteins or polypeptides of the presentinvention in combination with composites of biocompatible polymers, suchas, e.g., poly(lactic acid), poly(lactic-co-glycolic acid),methylcellulose, hyaluronic acid, collagen and the like. The structure,selection and use of degradable polymers in drug delivery vehicles havebeen reviewed in several publications, including A. Domb et al., 1992,Polymers for Advanced Technologies 3: 279-292, which is incorporatedherein by reference. Additional guidance in selecting and using polymersin pharmaceutical formulations can be found in the text by M. Chasin andR. Langer (eds), 1990, “Biodegradable Polymers as Drug Delivery Systems”in: Drugs and the Pharmaceutical Sciences, Vol. 45, M. Dekker, NY, whichis also incorporated herein by reference. Liposomes and liposomederivatives (e.g., cochleates, vesicles) can also be used to provide forthe sustained release of the antigen. In addition, methods formicroencapsulating antigens are well-known in the art, and includetechniques described, e.g., in U.S. Pat. Nos. 3,137,631; 3,959,457;4,205,060; 4,606,940; 4,744,933; 5,132,117; and International PatentPublication WO 95/28227, all of which are incorporated herein byreference.

[0133] In a non-limiting embodiment, the vaccine of the presentinvention can be a combination vaccine for protecting cats against FIVand, optionally, one or more other diseases or pathological conditionsthat can afflict felines, which combination vaccine comprises a firstcomponent comprising an immunologically effective amount of an antigenof the present invention selected from the group consisting of anFIV-related polynucleotide, protein or polypeptide as described above; asecond component comprising an immunologically effective amount of anantigen that is different from the antigen in the first component, andwhich is capable of inducing or enhancing the induction of a protectiveresponse against a disease or pathological condition that can afflictcats; and a veterinarily acceptable carrier.

[0134] The second component of the combination vaccine is selected basedon its ability to either induce or enhance the induction of a protectiveresponse against either FIV or another pathogen, disease, orpathological condition that afflicts cats, as known in the art, wherethe definition of the ability to induce or enhance the induction of aprotective response generally follows the definitions provided above asdirected to FIV, or as applied in parallel fashion to the otherpathogen, disease or pathological condition being treated, asappropriate. Any immunogenic composition now known or to be determinedin the future to be useful in a vaccine composition for cats can be usedas the second component of the combination vaccine. Such immunogeniccompositions include but are not limited to those that induce or enhancethe induction of a protective response against feline leukemia virus,feline herpes virus, feline calicivirus, or feline coronavirus, amongothers.

[0135] The antigen of the second component can optionally be covalentlylinked to the antigen of the first component to produce a chimericmolecule. In a non-limiting embodiment, the antigen of the secondcomponent comprises a hapten, the immunogenicity of which is detectablyincreased by conjugation to the antigen of the first component. Chimericmolecules comprising covalently linked antigens of the first and secondcomponents of the combination vaccine can be synthesized using one ormore techniques known in the art. For example, a chimeric molecule canbe produced synthetically using a commercially available peptidesynthesizer utilizing standard chemical synthetic processes (see, e.g.,Merrifield, 1985, Science 232:341-347). Alternatively, the separateantigens can be separately synthesized and then linked together by theuse of chemical linking groups, as known in the art. Alternatively, achimeric molecule can be produced using recombinant DNA technologywhereby, e.g., separate polynucleotide molecules having sequencesencoding the different antigens of the chimeric molecule are splicedtogether in-frame and expressed in a suitable transformed host cell forsubsequent isolation of the chimeric fusion polypeptide. Where thevaccine of the invention is a DNA vaccine, the spliced polynucleotidemolecule can itself be used in the vaccine composition, preferably inthe form of an expression vector. Ample guidance for carrying out suchrecombinant techniques is provided, among other places, in Maniatis etal., 1989, above; Ausubel et al., 1989, above; Sambrook et al., 1989,above; Innis et al., 1995, above; and Erlich, 1992, above.

[0136] The present invention further provides a method of preparing avaccine for protecting cats against FIV, comprising combining animmunologically effective amount of one or more antigens of the presentinvention selected from the group consisting of an FIV protein,homologous polypeptide, peptide fragment, fusion protein, analog andderivative, with a veterinarily acceptable carrier or diluent, in a formsuitable for administration to cats.

[0137] The present invention further provides a method of vaccinatingcats against FIV, comprising administering a vaccine compositioncomprising an immunologically effective amount of one or more antigensof the present invention selected from the group consisting of an FIVprotein, homologous polypeptide, peptide fragment, fusion protein,analog and derivative of the present invention to a cat. The amount ofFIV-related antigen administered will preferably range from about 0.1 μgto about 10 mg of polypeptide, more preferably from about 10 μg to about1 mg, and most preferably from about 25 μg to about 0.1 mg. In addition,the typical dose volume of the vaccine will range from about 0.5 ml toabout 5 ml per dose per animal.

[0138] Vaccine regimens can be determined as described above. Thevaccine can be administered in a single dose or in divided doses by anyappropriate route such as, e.g., by oral, intranasal, intramuscular,intra-lymph node, intradermal, intraperitoneal, subcutaneous, rectal orvaginal administration, or by a combination of routes. The skilledartisan will readily be able to formulate the vaccine compositionaccording to the route chosen. The timing of vaccination and the numberof boosters, if any, will preferably be determined by a veterinarianbased on analysis of all relevant factors, some of which are describedabove.

5.4. Oligonucleotide Molecules

[0139] The nucleotide sequences of the polynucleotide moleculesdisclosed herein provide the information necessary to constructoligonucleotide molecules that can be used as amplification primers andas probes in differential disease diagnosis, and these can readily bedesigned by the skilled artisan in light of this disclosure. Sucholigonucleotide molecules are preferably at least about 15 nucleotidesin length. Amplification of specific FIV genes and nucleotide sequencescan be carried out using such suitably designed oligonucleotides byapplying standard techniques such as, e.g., the polymerase chainreaction (PCR) which is described, among other places, in Innis et al.(eds), 1995, above; and Erlich (ed), 1992, above. In a preferredembodiment, such oligonucleotide molecules comprise nucleotide sequencesselected from the group consisting of SEQ ID NOS: 2 to 49, or thecomplements of said sequences.

[0140] Regarding diagnostics, oligonucleotide molecules of the presentinvention can be used in a standard amplification procedure to detectthe presence of an FIV polynucleotide molecule in a sample of felinetissue or fluid, such as brain tissue, lung tissue, placental tissue,blood, cerebrospinal fluid, mucous, urine, amniotic fluid, etc. Theproduction of a specific amplification product can be used to support adiagnosis of FIV infection, while lack of an amplified product may pointto a lack of infection.

[0141] Generally, for PCR amplification, a mixture comprising suitablydesigned primers, a template comprising the nucleotide sequence to beamplified, and appropriate PCR enzymes and buffers, is prepared andprocessed according to standard protocols to amplify a specific FIVpolynucleotide molecule of the template or a portion thereof. Otheramplification techniques known in the art, e.g., the ligase chainreaction, may alternatively be used.

[0142] The following examples are illustrative only, and are notintended to limit the scope of the present invention.

6. Example: Cloning of the FIV Proviral Genome and Individual Genes IntoEukaryotic Expression Vectors 6.1. Viral and Proviral DNA Isolation

[0143] Peripheral blood was obtained from a 5 yr old male cat prior toeuthanasia at the Capital Humane Society, Lincoln, Nebr. The plasmasample tested FIV positive and FeLV negative (FIV antibody and FeLVantigen kits, IDEXX, Westbrook, Me., USA), and the strain of FIV wasdesignated as FIV-141. The source FIV plasma sample was sterile-filtered(0.22 μM) and inoculated intravenously into an 8 wk old SPF cat(identification No. 96QGW2), which was then observed for 12 weeks.Peripheral blood samples were taken weekly. Tissue samples taken atnecropsy were used to confirm FIV infection, and to re-isolate andcharacterize the molecular and biological features of the new FIVisolate. Feline genomic DNA was isolated from the spleen of theFIV-inoculated SPF cat using a genomic DNA extraction kit (Stratagene,La Jolla, Calif.), and purified genomic DNA was dissolved in TE bufferat a concentration of 1 μg/μl and stored at −70° C.

6.2. Initial PCR Amplification and Cloning of Three Segments of theFIV-141 Genome

[0144] Three sets of oligonucleotides were designed based upon thepublished sequences of other FIV isolates (Talbott et al., 1989, Proc.Natl. Acad. Sci. USA, 86:5743-5747; Miyazawa et al., 1991, J. Virol.65:1572-1577; Talbott et al., 1990, J. Virol. 64:4605-4613). Theseoligonucleotides were used to amplify three segments of the FIV-141genome, the first segment at the 5′ end, the second segment at the 3′end, and the third segment in th middle of the genome. Because of a lowcopy number of the FIV proviral genome in infected tissue, two rounds ofPCR amplification were performed using a semi-nested set of primers foreach segment.

[0145] Three primers, designated as Pr-1 (SEQ ID NO:2), Pr-2 (SEQ IDNO:3) and Pr-8 (SEQ ID NO:8), were used to amplify the 5′ segment of theFIV-141 proviral genome comprising nucleotides 117 to 646 of SEQ IDNO:1. This segment spans the majority of the 5′ LTR, the interveningsequence between the 5′ LTR and the GAG open reading frame, and theN-terminal portion of the GAG gene. Primer sequences are presented inTABLE 1 below. The first round PCR amplification was performed using 200ng each of Pr-1 (SEQ ID NO:2) and Pr-8 (SEQ ID NO:8) as primers, and 1μg of feline genomic DNA as template, with a mixture of 0.5 U Taq DNApolymerase (Gibco BRL, Gaithersburg, Md.) and 1 U Pfu DNA polymerase(Stratagene, La Jolla, Calif.). PCR amplification conditions were: 94°C., 1 min; followed by 30 cycles of denaturing at 94° C. for 45 sec,annealing at 52° C. for 45 sec, and extension at 72° C. for 2 min. Thesecond round PCR amplification was performed using primers Pr-1 (SEQ IDNO:2) and Pr-2 (SEQ ID NO:3) (TABLE 1) and 2 μl of the first round PCRproducts as template. The same conditions as the first roundamplification were used except that the annealing temperature was 55° C.

[0146] Three primers, Pr-5 (SEQ ID NO:5), Pr-6 (SEQ ID NO:6) and Pr-7(SEQ ID NO:7) (TABLE 1), were used to perform two rounds of PCRamplifications and clone the 3′ segment of the FIV-141 proviral genome.The 3′ segment spans nucleotides 8874 to 9367 of SEQ ID NO:1, consistingof the intervening sequence between the 3′ LTR and the ENV gene, andmost of the 3′ LTR. Pr-5 (SEQ ID NO:5) and Pr-6 (SEQ ID NO:6) were usedin the first round, while Pr-6 (SEQ ID NO:6) and Pr-7 (SEQ ID NO:7) wereused for the second round PCR amplification. The same conditions wereapplied to the reaction and amplification in the first and second roundsas described above.

[0147] Three primers, Pr-3 (SEQ ID NO:4), Pr-9 (SEQ ID NO:9) and Pr-10(SEQ ID NO:10) (TABLE 1), were designed to amplify the segment fromnucleotides 5147 to 5631 of SEQ ID NO:1, spanning the C-terminal portionof the IN gene and the N-terminal portion of the Vif gene. The firstround amplification was performed using Pr-3 (SEQ ID NO:4) and Pr-10(SEQ ID NO:10), followed by the second round amplification using Pr-9(SEQ ID NO:9) and Pr-10 (SEQ ID NO:10). The same conditions were appliedto the reaction and amplification in the first and second rounds asdescribed above.

[0148] PCR products from each of the three second round amplificationswere applied to a 1% agarose gel and the expected size bands for allthree regions were purified (Wizard PCR Preps kit; Promega, Madison,Wis.). The purified PCR fragments were cloned into pCR-Script Amp SK(+)vector (Stratagene, La Jolla, Calif.) according to manufacturer'sinstructions, and their presence was confirmed by restriction enzymedigestion. The FIV specific sequence of each clone was determined bysequencing the two strands of the plasmid DNA (Advanced Genetic AnalysisCenter, St. Paul, Minn.). The consensus sequence from three independentclones was used to define the authentic FIV-141 sequence. TABLE 1Primers us d for cl ning th FIV-141 pr viral genom from infected catspleen genomic DNA Primer (SEQ ID NO:) Direction Sequence Pr-1 (2)forward 117-CCGCAAAACCACATCCTATGTAAAGCTTGC- 146 Pr-2 (3) reverse646-CGCCCCTGTCCATTCCCCATGTTGCTGTAG-61 7 Pr-3 (4) forward4738-ACAAACAGATAATGGACCMATTTTAAAAA-4767 Pr-5 (5) forward8793-GCAATGTGGCATGTCTGAAAAAGAGGAGGA-8822 Pr-6 (6) reverse9367-TCTGTGGGAGCCTCMGGGAGAACTC-9342 Pr-7 (7) forward8874-TCTTCCCTTTGAGGMGATATGTCATATGAATCC-8907 Pr-8 (8) reverse1047-TTACTGTTTGAATAGGATATGCCTGTGGAG-1 018 Pr-9 (9) forward5147-TTAAAGGATGAAGAGAAGGGATATTTTCTT-5 176 Pr-10 (10) reverse5631-TTTCAATATCATCCCACATMATCCTGT-5604 Pr-11 (11) forward1-TGGGAAGATTATTGGGATCCTGAAGAAATA-30 Pr-12 (12) reverse5460-CATATCCTATATAATAATCACGCGTATGAAAG- CTCCACCT-5421 Pr-13 (13) forward5421-AGGTGGAGCTTTCATACGCGTGATTATTATAT AGGATATG-5460 Pr-14 (14) reverse9464-TGCGAGGTCCCTGGCCCGGACTCC-9441 Pr-16 (15) reverse9444-CTCCAGGGATTCGCAGGTAAGAGAAATTA-9416

[0149] Combination of the sequences from the 5′ and 3′ end segmentsrevealed that the LTR of FIV-141 consists of 354 bases, including 208bases in the U3 region (from nt 1 to 208), 79 bases in the R region(from nt 209-287), and 67 bases in the U5 region (from nt 288 to 354).The terminal 2-base inverted repeat, the TATA box, the polyadenylationsignal, and a number of putative cis-acting enhancer-promoter elementswere perfectly conserved when compared with other FIV isolates (Talbottet al, 1989, Proc. Natl. Acad. Sci. 86:5743-5747; Miyazawa et al., 1991,J. Virol. 65:1572-1577; Talbott et al., 1990, J. Virol. 64:4605-4613).

6.3. PCR Amplification and Cloning of the Entire Proviral Genome ofFIV-141

[0150] The sequence information obtained from the three segmentsdescribed above was used to design FIV-141-specific primers to amplifyand clone the entire proviral genome in two pieces, the 5′ half and 3′half. Each half was amplified in two rounds of PCR amplification with asemi-nested set of primers. The 5′ half of the genome (nts 1 to 5460 ofSEQ ID NO:1) was amplified as follows. For the first round PCRamplification, primers Pr-11 (SEQ ID NO:11) and Pr-10 (SEQ ID NO:10)were used (TABLE 1). Briefly, the PCR reaction was set up in a totalvolume of 50 μl, containing 1 μl of feline genomic DNA template (1μg/μl), 1 μl of each primer (100 ng/μl), 5 μl of 10×Advantage Tth PCRreaction buffer (Advantage Genomic PCR Kit; Clontech, Palo Alto,Calif.), 2.2 μl of 25 mM Mg(Oac)₂, 1 μl of 50×dNTP mix (10 mM each), 1μl of 50×Advantage Tth Polymerase mix, 1 μl of Pfu polymerase (2.5U/μl), and 36.8 μl of sterile water. The reaction mix was heated at 94°C. for 2 min, followed by 30 cycles of amplification; 94° C. for 30 secand 68° C. for 6 min. The second round PCR amplification was carried outusing 2 μl of the first round PCR product as template and primers Pr-11(SEQ ID NO:11) and Pr-12 (SEQ ID NO:12) (TABLE 1). To facilitate thelater construction of a full-length FIV-141 clone, an Mlu I restrictionsite was incorporated into primer Pr-12 (SEQ ID NO:12) by a silentmutation. The Mlu I site is underlined in the Pr-12 (SEQ ID NO:12)primer sequence (TABLE 1). The same PCR conditions were used in thesecond round. After two PCR amplification rounds, a 5460 bp fragment wasobtained.

[0151] To clone the 3′ half of the proviral genome of FIV-141, threeprimers, Pr-9 (SEQ ID NO:9), Pr-13 (SEQ ID NO:13) and Pr-14 (SEQ IDNO:14), were initially used (TABLE 1). The first round PCR amplificationwas carried out using Pr-9 (SEQ ID NO:9) and Pr-14 (SEQ ID NO:14). Pr-14(SEQ ID NO:14) is a primer composed of the last 24 bases at the 3′ endof the FIV-141 proviral genome. The second round amplification wasperformed using primers Pr-13 (SEQ ID NO:13) and Pr-14 (SEQ ID NO:14).Pr-13 (SEQ ID NO:13) was overlapped with Pr-12 (SEQ ID NO:12). As withPr-12 (SEQ ID NO:12), an Mlu I restriction site was incorporated intoPr-13 (SEQ ID NO:13) to facilitate the later construction of thefull-length FIV clone. Unfortunately, after two rounds of PCR, nospecific DNA band was amplified. Failure to amplify the 3′ half genomemay have been due to a high GC content and very stable secondarystructure in primer Pr-14 (SEQ ID NO:14). Therefore, a new primer, Pr-16(SEQ ID NO:15), was designed with a sequence ending 20 bases upstream ofthe last base in the genome. First round PCR amplification was performedusing primers Pr-9 (SEQ ID NO:9) and Pr-16 (SEQ ID NO:15), followed by asecond round amplification using primers Pr-13 (SEQ ID NO:13) and Pr-16(SEQ ID NO:15). A DNA fragment of the expected size was obtained afterthe second round amplification.

[0152] The DNA fragments of the 5′ half and 3′ half of the FIV-141genome were purified (Wizard PCR Preps DNA purification kit; Promega,Madison, Wis.) and cloned into pCR-Script Amp SK(+) cloning vector(Stratagene, La Jolla, Calif.). Clones from three independent PCRreactions were sequenced for each of the 5′ half and 3′ half clones. Thesequences were obtained by sequencing both strands of the plasmid DNA(Advanced Genetic Analysis Center, St. Paul, Minn.). The authenticconsensus sequence for the entire genome of FIV-141 was obtained bycombining the sequences of the three independent clones, and ispresented herein as SEQ ID NO:1. The DNAStar program (DNAStar Inc.,Madison, Wis.) was used to perform sequence assembly, comparison andanalysis.

6.4. Cloning of Individual FIV-141 Genes Into pCMV-MCS and pCMV-HAEukaryotic Expression Vectors

[0153] The pCMV-MCS expression vector (FIG. 2) was constructed from thepCMVβ reporter vector (Clontech, Palo Alto, Calif.) in the followingsteps. The β-galactosidase gene was removed from the reporter vectorusing the Not I restriction sites. A synthetic DNA fragment containingmultiple cloning sites (MCS) including a Not I site at both ends, andEcoR V, Avr II, Bgl II, Cla I, Kpn I, Nru I, Pac I, Nhe I, Swa I, Apa I,Sma I, and Spe I sites, was inserted into the backbone pCMVβ vector togenerate the expression vector pCMV-MCS. To construct the pCMV-HAexpression vector (FIG. 3), a synthetic DNA fragment consisting of asequence encoding a nine amino acid epitope tag from human influenzahemagglutinin (HA) protein and a translation stop codon immediatelyfollowing the HA tag was inserted into the pCMV-MCS vector at the Apa Iand Spe I sites.

[0154] Four FIV genes, including ENV, SU, GAG, and POL, were separatelycloned into the pCMV-MCS vector in a similar fashion. Briefly, twoprimers were designed to amplify the particular gene using either the 5′half or 3′ half FIV-141 clones as template (TABLE 2). A Cla I and a SpeI restriction enzyme site were incorporated into the forward and reverseprimers, respectively, to facilitate cloning into the vector. The PCRreaction was set up in a volume of 100 μl, consisting of 10×reactionbuffer (10 μl), 50 mM MgCl₂ (3 μl), 10 mM dNTP (2 μl), each primer (1μl, 100 ng/μl), template (1 μl, 1 μl), Taq DNA polymerase (0.5 μl, 5U/μl), and sterile H₂O (81.5 μl). PCR amplifications were performed asfollows: 94° C. for 3 min; followed by 30 cycles of 94° C. for 30 sec,55° C. to 62° C. for 2 min. The PCR fragment was purified using a WizardPCR prep DNA purification kit (Promega, Madison, Wis.), digested withCla I and Spe I, and then inserted into the pCMV-MCS expression vectorthat had been digested with these same two restriction enzymes. Theentire open reading frame of each gene was verified by sequencing.

[0155] For cloning the ENV gene, forward and reverse primers were used,as shown in TABLE 2 below. The Cla I site in the forward primer and theSpe I site in the reverse primer are underlined.

[0156] For cloning the SU gene, forward and reverse primers were used,as shown in TABLE 2. The two restriction enzyme sites are underlined. Astop codon (TCA) was incorporated in the reverse primer at nts 23-25.

[0157] For cloning the GAG gene, forward and reverse primers were used,as shown in TABLE 2. The Cla I site in the forward primer and the Spe Isite in the reverse primer are underlined. TABLE 2 Primer sequences usedto clone FIV genes into eukaryotic ex- pression vectors.^(a) ForwardPrimer Reverse Primer Expression Vector Gene (SEQ ID NO.) (SEQ ID NO.)pCMV-MCS ENV 5′-TTTCATCTGCATCGA 5′-CCTGTATTCTACTAGT TAAACATGGCGGAGGGCTGAAATGCTCCATCAT-3′ AGG-3′ (16) (17) pCMV-MCS SU 5′-TTTCATCTGCATCGAT5′-GGGCTAACATMTATGA AAACATGGCGGAGGGA CTAGTTCACCTTTTTTGTT GG-3′ (18)TACCTTTATACCT-3′ (19) pCMV-MCS GAG 5′-GGTAGGATCGATTC5′-GTCTTCACTAGTAAGTT TACAGCAACATGGGGA GTGGTAGTACCCATTGTA ATGG-3′ (20)TTATAGT-3′ (21) pCMV-MCS POL I 5′-GGTAAAAATCGATAAT 5′-ACTGCAACTAGTCTTCTTGAAACGGGGCGATGG ACTTACCTGCCAATCTTC GGCGAGC-3′ (22) G-3′ (23) pCMV-MCSPOLII 5′-TGTTAGATCGATAAT 5′-ACTGCAACTAGTCTTC GTATAATAAAGTGGGTATACTTACCTGCCAATCTTC CCACC-3′ (24) G-3′ (25) pCMV-HA MA5′-GGTAGGATCGATTCT 5′-CTGTTTGGGGCCCAT ACAGCMCATGGGGAAT AAGCCTGTGGAGGTCCTGG-3′ (26) TCTTC-3′ (27) pCMV-HA CA 5′-GGACCTATCGATACC5′-TGCTTGGGGCCCTTGC ATGCCTATTCAAACAGT ACCCTAGTAAGAGCCTCT AAATGGAGCACC-3′(28) GC-3′ (29) pCMV-HA NC 5′-GGCTCTTATCGATAC 5′-TATTATGGGCCCCATACATGACAGTTCAAGCAA TCTAACAATTTCTCCTCTA AAGGACCAAG-3′ (30) CCG-3′ (31)pCMV-HA Vif 5′-CCCTGCACTCTTCAT 5′-GGGATTATTTCTTCGG CGATACCATGAGTGACGGCCCTAATTCTCCTGTCC AAGATTGGCAGG-3′ (32) ACAATAAATTCCT-3′ (33) pCMV-HAORF-2 5′-TTGTGGACGGGAATC 5′-ATATTAAAAGAAATAGG GATACCATGGAAGAAATGCCCGGCAGTATTTATGG AATCCCACTG-3′ (34) ATAATGT-3′ (35) pCMV-HA TM5′-GTATAAAGGTATCGA 5′-TGAAATGCTGGGCCCT TACCATGGCCGCTATTCTCCTCCTCTTTTTCAGATA ATATTATGTTAGCC-3′ TGCCACA-3′ (37) (36) pCMV-HA Rev5′-TTTCATCTGCATCG 5′-TGTACGGGGCCCGTCC ATAAACATGGCGGAGGATTAGCATTTTTTCTATTT GAGG-3′ (38) C-3′ (39) pCMV-HA PR5′-TGTTAGATCGATAATG 5′-CTCTGAAACGGGCCCC TATAATAAAGTGGGTACATTACCAACCTTATGTTGA CACC-3′ (40) ACTTAATC-3′ (41) pCMV-HA RT5′-AACATAATCGATACCA 5′-TTCTAGGGGCCCCATC TGGTCCAGATTTCAGAGGTTTGACAAAGTTCATCTA AAAATTCCAATAG-3′ (42) CCTC-3′ (43) pCMV-HA DU5′-CTTTGTATCGATACCA 5′-CACCCAGGGCCCAAAG TGGTTATAGAAGGTGAAACTCCAGTTGACCCAAAT GGAATATTAG-3′ (44) CCC-3′ (45) pCMV-HA IN5′-GGGTCAATCGATACA 5′-CAATCTGGGCCCCTCA ATGTCTTCATGGGTGGATCACCTTCAGGAAGAGTG CAGAATTGAA-3′ (46) CAGG-3′ (47)

[0158] For cloning the POL gene into the vector, two expressionconstructs (POL I and POL II) were made. The N-terminus of the POL Iconstruct begins with the first residue of the shifted POL open readingframe, while the N-terminus of the POL II construct begins at the firstresidue of the protease (PR) protein. An initiation (ATG) codon withKozak context was introduced immediately upstream of the POL gene forboth POL I and POL II constructs. For cloning POL I, forward and reverseprimers were used as shown in TABLE 2. The two restriction sites areunderlined, and the introduced ATG initiation codon in the forwardprimer is at primer nts 15-17. For cloning POL II, forward and reverseprimers were used as shown in TABLE 2. The two restriction sites areunderlined, and the introduced ATG initiation codon in the forwardprimer is at primer nts 14-16.

[0159] Eleven FIV-141 genes, including matrix (MA), capsid (CA),nucleocapsid (NC), protease (PR), reverse transcriptase (RT),deoxyuridine triphosphatase (DU), integrase (IN), transmembrane (TM),Vif, ORF2 and Rev, were each separately cloned into the expressionvector, pCMV-HA. All the clones, except for Rev, were made in a similarfashion. Each individual gene was amplified using either the 5′ half or3′ half clone of FIV-141 as template, and a set of primers as shown inTABLE 2, with the same amplification conditions as above. Cla I and ApaI restriction sites were incorporated into forward and reverse primers,respectively. After being digested with Cla I and Apa I, each genefragment was cloned into the pCMV-HA expression vector that had beendigested with these same two restriction enzymes. To clone the Rev gene,total mRNA extracted from FIV-141-infected peripheral blood mononuclearcells (PBMCs) was reverse transcribed into cDNA, followed by PCRamplification. For each clone, the entire open reading frame of eachgene was verified by sequencing.

[0160] For cloning MA, forward and reverse primers were used as shown inTABLE 2. The introduced Cla I and Apa I sites are underlined in theforward and reverse primer, respectively.

[0161] For cloning CA, forward and reverse primers were used as shown inTABLE 2. An initiation codon (ATG) was incorporated into the forwardprimer immediately upstream of the coding region of the CA gene, asshown in TABLE 2 at primer nts 16-18. The introduced Cla I and Apa Isites in the forward and reverse primers, respectively, are underlined.

[0162] For cloning NC, forward and reverse primers were used as shown inTABLE 2. An initiation codon (ATG) was incorporated into the forwardprimer immediately upstream of the coding region of the NC gene, asshown in TABLE 2 at primer nts 17-19. The introduced Cla I and Apa Isites in the forward and revers primers, respectively, are underlined.

[0163] For cloning Vif, forward and reverse primers were used as shownin TABLE 2. The introduced Cla I and Apa I sites in the forward andreverse primers, respectively, are underlined.

[0164] For cloning ORF2, forward and reverse primers were used as shownin TABLE 2. The introduced Cla I and Apa I sites in the forward andreverse primers, respectively, are underlined.

[0165] For cloning Rev, forward and reverse primers were used as shownin TABLE 2. The ATG initiation codon in the primer at nts 21-23 is theauthentic ATG codon from Rev. The introduced Cla I and Apa I sites inthe forward and reverse primer, respectively, are underlined.

[0166] For cloning TM, forward and reverse primers were used as shown inTABLE 2. An initiation codon (ATG) was incorporated in the forwardprimer immediately upstream of the coding region of the TM gene, asshown in TABLE 2 at primer nts 20-22. The introduced Cla I and Apa Isites in the forward and reverse primer, respectively, are underlined.

[0167] For cloning PR, forward and reverse primers were used as shown inTABLE 2. An initiation codon (ATG) was incorporated in the forwardprimer immediately upstream of the coding region of the PR gene, asshown in TABLE 2 at primer nts 14-16. The introduced Cla I and Apa Isites in the forward and reverse primers, respectively, are underlined.

[0168] For cloning RT, forward and reverse primers were used as shown inTABLE 2. An initiation codon (ATG) was incorporated in the forwardprimer immediately upstream of the coding region of the RT gene, asshown in TABLE 2 at primer nts 16-18. The introduced Cla I and Apa Isites in the forward and reverse primers, respectively, are underlined.

[0169] For cloning DU, forward and reverse primers were used as shown inTABLE 2. An initiation codon (ATG) was incorporated in the forwardprimer immediately upstream of the coding region of the DU gene, asshown in TABLE 2 at primer nts 16-18. The introduced Cla I and Apa Isites in the forward and reverse primers, respectively, are underlined.

[0170] For cloning IN, forward and reverse primers were used as shown inTABLE 2. An initiation codon (ATG) was incorporated in the forwardprimer immediately upstream of the coding region of the IN gene, asshown in TABLE 2 at primer nts 16-18. The introduced Cla I and Apa Isites in the forward and reverse primers, respectively, are underlined.

7. Example: in Vitro Expression of FIV Genes 7.1. Transfection of FIVGene Constructs

[0171] Plasmid DNAs from each of the FIV gene constructs prepared asdescribed above were separately transfected (2 μg/well) into Crandellfeline kidney (CRFK) cells that were 40-60% confluent in a 6-well plate,using Trans IT Polyamine Transfection Reagents (Mirus, Madison, Wis.)according to the manufacturer's protocol. Briefly, Trans IT Lt-1(PANVERA) (10 μl) was mixed with RPMI 1640 medium (1 ml) and incubatedat room temperature for 15 min. Plasmid DNA (2 μg) was added to theRPMI/Lt-1 solution, and incubated at room temperature for another 15min. Old culture medium was removed from the cell monolayer, and thecells were washed once with serum-free RPMI. The cells were thenincubated in the DNA cocktail in a CO₂ incubator (5% CO₂) at 37° C. for4 hr. The DNA cocktail was removed, and 2 ml of fresh RPMI 1640 mediumwith 3% fetal calf serum (FCS) was added to each well. Transfected cellswere analyzed 48 hr later for protein expression by Western blot,RT-PCR, and functional RT activity assays.

7.2. FIV-141 Gene Expression Detected by Western Blot Analysis

[0172] Transfected CRFK cells prepared as described above were harvestedat 48 hr and lysed with 2×SDS-PAGE loading buffer. Cell lysates wereloaded and run on a 4-20% gradient SDS-PAGE gel, followed by proteintransfer onto polyvinylidene difluoride (PVDF) membranes (0.22 μM,BIO-RAD, Hercules, Calif.). The-primary antibody, anti-HA MAb specificto the HA tag (Boehringer Mannheim, Indianapolis, Ind.), or serum fromFIV infected cats, was applied to the appropriate blot and incubated for3 hr. After washing, antibody binding to specific proteins was detectedwith an alkaline phosphate-conjugated secondary antibody, as recommendedby the manufacturer (Boehringer Mannheim, Indianapolis, Ind.).

[0173] A specific protein band of the appropriate size was detected byWestern blot for eleven expression clones, including the GAGpolyprotein, MA, CA, NC, PR, DU, IN, Vif, Rev, ORF₂ and TM, indicatingthat transfected cells carrying these constructs expressed thecorresponding FIV gene products.

7.3. FIV-141 Gene Expression Detected by RT-PCR

[0174] Seven expression constructs, including PR, DU, ENV, SU, Vif, POLI and POL II, were assayed by RT-PCR. Total RNA was harvested fromtransfected CRFK cells at 48 hr by lysing with a tissue shredder(Qiagen, Chatsworth, Calif.), followed by purification (RNA purificationkit; Qiagen). RNA was eluted in DEPC-treated H₂O (50 μl). cDNA was madewith random hexamers as primers and SuperScript II reverse transcriptase(Gibco BRL, Gaithersburg, Md.). Duplicate reactions were set up in thepresence or absence of SuperScript II RT. FIV specific oligonucleotideswere used to amplify gene specific fragments by PCR. PCR products wereanalyzed by running each sample (10 μl) on a 1.2% agarose gel.

[0175] The RT-PCR data indicated that cells transiently transfected witheach of the seven constructs expressed the appropriate mRNA.

7.4. FIV-141 Gene Expression Detected by Reverse Transcriptase (RT)Activity Assay

[0176] Gene expression of the RT, POL I and POL II constructs was testedby RT activity assay using a Reverse Transcription Assay Kit (BoehringerMannheim). Transfected CRFK cells were harvested at 48 hrpost-transfection, and resuspended in lysis buffer (40 μl). The RT ELISAassay was performed according to the manufacturer's protocol (BoehringerMannheim, Indianapolis, Ind.). The results showed that the RT construct,but not the POL I and POL II constructs, resulted in significant RTactivity, indicating that functional RT protein was expressed intransfected cells carrying the RT construct. Failure to detect RTactivity for the POL I and POL II constructs may indicate that theexpressed protein was not properly processed.

8. Example: Efficacy of DNA Vaccination in Cats 8.1. Preparation ofPlasmid DNA for Vaccination

[0177] Production of bulk purified plasmid DNA for this animal study wascontracted to Bayou Biolabs (Harahan, La.). Coded samples of plasmid DNA(1 μg) were sent and each was transformed into DH5α E. coli competentcells. Each clone was grown in 8 liters of enriched broth. Plasmid DNAin all forms was purified first, followed by purification of thesupercoiled form. The final purified DNA was dissolved in phosphatebuffered saline (PBS) with 1 mM EDTA at a concentration of 2-5 μg/μl.DNA concentration was determined by UV absorbance and by fluorometry.Both methods gave the same concentration, confirming an absence ofnucleotide contamination. Plasmid DNA was also analyzed byelectrophoresis on a 1% agarose gel. Ethidium bromide stained gelsshowed that all the supercoiled purified plasmid DNA preparations were amixture of mostly supercoiled plasmid with a small amount of dimericsupercoiled plasmid. No linear or nicked plasmid DNA, chromosomal DNA ormRNA contamination was visible on the gel. The plasmid DNA was shippedon dry ice and stored at −70° C.

8.2. Vaccination and Challenge

[0178] Four experimental vaccines were assembled, each with differentcombinations of the 16 FIV-141 gene constructs (300 μg DNA from eachconstruct/dose). Vaccine 1 (XFIV-1) consisted of all 16 FIV-141 geneconstructs. Vaccine 2 (XFIV-2) consisted of 7 structural geneconstructs, including GAG, MA, CA, NC, ENV, SU and TM. Vaccine 3(XFIV-3) consisted of a mixture of structural and nonstructural geneconstructs, including DU, PR, GAG, NC and MA. Vaccine 4 (XFIV-4)consisted of 9 nonstructural and regulatory gene constructs, includingRev, ORF2, Vif, IN, DU, RT, PR, POL I and POL II.

[0179] Placebo consisted of both pCMV-MCS and pCMV-HA expression vectorsin equal proportions. Since the total DNA given in one dose variedbetween experimental vaccines, the amount of placebo DNA (2400 μg) wasan average between the highest and lowest DNA doses. The appropriatevolume of stock DNA from each construct was dissolved in sterile PBS(GIBCO). The final volume for each dose ranged from 2-3 ml.

[0180] Antibody-profile defined, barrier-reared, domestic cats (n=60,approximately 8 weeks of age, no smaller than 0.5 kg) were obtained fromLiberty Research, Inc. (Waverly, N.Y.). Cats were vaccinated with killedvaccines to feline herpes virus, feline calicivirus, and felineparvovirus. Sixty cats were randomly assigned by litter and sex to 6groups prior to vaccination (TABLE 3). TABLE 3 GROUP VACCINE CHALLENGE n1 Placebo YES 10 2 Placebo NO 10 3 XFIV-1 YES 10 4 XFIV-2 YES 10 5XFIV-3 YES 10 6 XFIV-4 YES 10

[0181] Combinations and doses of FIV-141 gene constructs in experimentalvaccines are detailed in TABLE 4. TABLE 4 VACCINEANTIGEN(S)-QUANTITY/DOSE VOL/DOSE Placebo 1200 μg pCMV-MCS DNA + 1200 μg2 ml pCMV-HA DNA; Total = 2400 μg DNA XFIV-1 300 μg each DNA from Gag,POL I, POL II, 3 ml ENV, SU, TM, PR, RT, DU, IN, Ma, CA, NC, Vif, Rev,ORF2; Total = 4800 μg DNA XFIV-2 300 μg each from Gag, TM, SU, ENV, NC,2 ml CA, MA; Total = 2100 μg DNA XFIV-3 300 μg each of DU, PR, NC, Gag,MA; 2 ml Total = 1500 μg DNA XFIV-4 300 μg each of Rev, ORF2, Vif, IN,DU, 2 ml RT, PR, POL I, POL II; Total = 2700 μg

[0182] Vaccines were administered intramuscularly at 4 week intervalswhen cats were 8, 12 and 16 weeks of age. Four weeks following the lastvaccination, cats were challenged at age 20 weeks by inoculating 354TCID₅₀ FIV-141 virus subcutaneously in the nape of the neck, and wereobserved for a total of 8 weeks post-challenge.

8.3. Evaluation of Vaccine Efficacy

[0183] Similar to HIV-1 disease progression (Graziosi et al., 1993,Proc. Natl. Acad. Sci. 90:6405-6409), FIV RNA load in plasma has beendemonstrated to correlate with disease stage, and can predict diseaseprogression in accelerated FIV infection (Diehl et al., 1995, J. Virol.69:2328-2332; Diehl et al., 1996, J. Virol. 70:2503-2507). In thisstudy, peripheral blood was drawn weekly to monitor the efficacy of thevaccination. Plasma viral loads were determined by both quantitativecompetitive-reverse transcription-polymerase chain reaction (QcRT-PCR),and quantitative virus isolation on FeP2 cell culture.

8.3.1. Quantitation of Viral RNA in Plasma by QcRT-PCR

[0184] Viral RNA was isolated from plasma samples using QlAmp Viral RNAPurification Kit (Qiagen). Each purified RNA sample was distributed intofour tubes, and into each tube was added an internal competitive RNAtemplate with decreasing amounts of RNA (from 1000 fg, 100 fg, 10 fg to1 fg). RNA samples were subjected to RT-PCR using the Titan One TubeRT-PCR System (Boehringer Mannheim). A one-step PCR protocol provided bythe manufacturer was performed with minor modifications to increase thesensitivity of the assay. The RT-PCR reaction was set up in a totalvolume of 38.5 μl containing: 6.5 mM DTT, 0.3 units RNase inhibitor, 0.3mM dATP, 0.3 mM dGTP, 0.3 mM dTTP, 0.3 mM dCTP, 10.4 ng of each FIVspecific oligonucleotide, i.e., QPCR-1 (forward primer1392-TGTAGAGCATGGTAT CTTGMGCATTAGGAAA-1423) (SEQ ID NO. 48), and QPCR-O2(reverse primer 2175-GTTCCTCTCTTTCCGCCTCCTACTCCMTCATATT-2141) (SEQ IDNO:49), 1.95 mM MgCl₂, and 1 μl of Titan Enzyme Mix. RT-PCRamplification conditions were 50° C. for 90 min, 94° C. for 3 min;followed by 30 cycles of denaturing at 94° C. for 30 sec, annealing at55° C. for 1 min, and extension at 72° C. for 2 min; followed by 72° C.for 10 min.

[0185] Each PCR sample was separated on a 1.0% agarose gel and stainedwith ethidium bromide. Quantitation of viral RNA load was determined bycomparing the intensity of the positive DNA band with that of theinternal competitive standard control DNA band using the Gel-Doc system(Bio-Rad Laboratories).

8.3.2. Quantitation of Viral Load in Plasma by Virus Isolation

[0186] Virus quantitation by culture was performed based on amodification of a method described by Meers et al., 1992, Arch. Virol.127:233-243. An IL-2 dependent feline T cell line (FeP2 cells) developedin the laboratory was used for virus isolation from plasma. FeP2 cellswere grown in complete medium (CM) consisting of RPMI 1640 supplementedwith 10% heat-inactivated fetal bovine serum (FBS), 1% GlutaMAX-1,Insulin-Transferrin-Selenium-S (ITS-S) 43 mg/ml (100×) at 1%,Non-Essential Amino Acids Solution (NEAA) 10 mM (100×) at 1%,2-mercaptoethanol 5.5×10⁻² M (1000×) at 1 μl/ml, sodium pyruvate 100 mM(100×) at 1%, gentamicin 50 mg/ml at 0.1%, recombinant human IL-2 at 100U/ml, and Con A at 1 μg/ml. Each plasma sample was diluted 10-fold (10⁻¹to 10⁻⁴) in a 48-well plate using diluent media (DM) consisting ofRPMI-1640 supplemented with 5% FBS, 20 mM HEPES, 50 μg/ml gentamicin, 4U/ml heparin. Infection was carried out by adding 8.5×10⁵ of FeP2 cellsinto each well, followed by incubating at 37° C. for 0.5 to 2 hr. CM(700 μl) was then added to each well and cultures were maintained in thefollowing manner: at day 3, 500 μl of supernatant was removed from eachwell and 700 μl of CM was added back; at days 7, 10, 13, 16, 19, 22 and25, 675 μl of supernatant was removed from each well and 700 μl of CMwas added back. Cultures were terminated at day 30. From day 19 to 30,the culture fluid removed from each well was tested for the presence ofFIV p26 using an FIV p26 antigen kit (IDEXX). Virus titer was calculatedas the reciprocal of the last positive dilution and reported as tissueculture infectious dose (TCID)₁₀₀/mL.

8.3.3. Viral Load in Plasma Post-Challenge

[0187] Compared with group 1 (placebo, challenged), groups 4 and 5exhibited significant decreases in cumulative plasma viral load duringthe period of 8 weeks post-challenge. Groups 4 and 5 exhibited decreasesof 19.3 fold and 25.4 fold, respectively, in cumulative plasma viralloads, as determined by virus isolation (FIG. 4). Groups 4 and 5 alsoexhibited decreases of 7.0 and 7.1 fold, respectively, in cumulativeplasma viral RNA loads, as detected by QcRT-PCR (FIG. 5). Group 4 wasvaccinated with XFIV-2 consisting of 7 structural gene constructs,including GAG, MA, CA, NC, ENV, SU and TM. Group 5 was vaccinated withXFIV-3 consisting of a mixture of structural and nonstructural geneconstructs, including GAG, NC, MA, DU and PR. Group 3 exhibited a 4.4fold decrease in plasma viral load, as determined by virus isolation,and a decrease of 6.7 fold in plasma viral RNA load, as determined byQcRT-PCR (FIGS. 4, 5). Group 3 was vaccinated with XFIV-1 consisting ofall 16 gene constructs. Group 6 exhibited a 3.8 fold decrease in plasmaviral load and plasma viral RNA load, as determined by virus isolationand QcRT-PCR, respectively (FIGS. 4, 5). Group 6 was vaccinated withXFIV-4 consisting of 9 nonstructural and regulatory gene constructs,including Rev, ORF2, Vif, IN, DU, RT, PR, POL I and POL II.

[0188] Compared with group 1 (placebo, challenged), group 5 exhibitedfewer time points when plasma virus titers were higher than 10infectious doses/ml, as detected by virus isolation (FIG. 6). Throughoutthe 8 week study, only 3 cats, for a total of 4 time points, weredetermined to have virus titers higher than 10 infectious doses/ml.Consistent with this observation is the decreased number of totalpositive time points in group 5, as detected by QcRT-PCR (FIG. 7). ViralRNA in plasma was detected at 10 time points throughout the whole studyfor group 5. Following group 5 is group 4, which had a marked decreasein the number of positive time points at which virus titers were higherthan 10 infectious doses/ml, as detected by virus isolation. Moderatelyfewer positive time points, compared to control, for plasma viral RNAwere detected in group 4 by QcRT-PCR throughout the whole study (FIGS. 6and 7). A slight decrease in positive time points for both virusisolation and QcRT-PCR was observed in group 3 compared with positivecontrol (FIGS. 6 and 7). There appears to be no difference between group6 and group 1 in terms of the number of positive time points detected byboth virus isolation and QcRT-PCR (FIGS. 6 and 7).

[0189] Although no vaccinated group tested in this study was totallyprotected from infection (i.e., sterilizing immunity), decreases inplasma viral load were demonstrated in groups 4 and 5 during themonitoring period of 8 weeks post-challenge. FIV antigens in commonamong the two groups as encoded by the vaccine DNA include the GAGpolyprotein, MA and NC proteins. Among the various conclusions of thisstudy, we conclude that the GAG polyprotein and its substituentproteins, MA, CA and NC can stimulate protective immunity against FIVinfection, and that polynucleotide molecules encoding these proteins areuseful as vaccine agents.

[0190] All patents, patent applications, and publications cited aboveare incorporated herein by reference in their entirety.

[0191] The present invention is not limited in scope by the specificembodiments described, which are intended as single illustrations ofindividual aspects of the invention. Functionally equivalentcompositions and methods are within the scope of the invention. Indeed,various modifications of the invention, in addition to those shown anddescribed herein, will become apparent to those skilled in the art fromthe foregoing description. Such modifications are intended to fallwithin the scope of the appended claims.

1 52 1 9464 DNA Feline immunodeficiency virus 1 tgggaagatt attgggatcctgaagaaata gaaaaaatgc taatggactg aggacgtaca 60 taaacaagtg acagatggaaacagctgaat atgactcaat gctagcagct gcttaaccgc 120 aaaaccacat cctatgtaaagcttgccgat gacgtgtatc ttgctccatt ataagagtat 180 ataaccagtg ttttgtaaaagcttcgagga gtctctctgt tgagggcttt cgagttctcc 240 cttgaggctc ccacagatacaataaaaaac tgagctttga gattgaaccc tgtcttgtat 300 ctgtgtaatt tctcttacctgcgaatccct ggagtccggg ccagggacct cgcagttggc 360 gcccgaacag ggacttgaaaaggagtgatt agggaagtga agctagagca atagaaagct 420 gtcaagcaga actcctgcaggccttgtatg gggagcagtt gcagacgctg ctggcagtga 480 gtatctctag tggagcggacctgagctctg gattaagtca ctgctcacag gcctagataa 540 agattatctg gtgactcttcgcggatcgtc aaaccagggg attcgtcggg ggacagccaa 600 caaggtagga gagattctacagcaacatgg ggaatggaca ggggcgagac tggaaaatgg 660 ccattaagag atgtagtaatgttgctgtag gggtagggag caggagtaaa aaatttggag 720 aaggaaattt tagatgggccataaggatgg ctaatgtaac tacaggacga gaacctggtg 780 atataccaga gactttagaacagctaagat caatcatttg tgacttacaa gacagaagag 840 aacaatatgg atctagtaaagaaattgaca tggcaattac cactttaaaa gtttttgcag 900 tggcaggaat tctaaatatgactgtaacta ctgccacagc agctgaaaat atgtatgctc 960 agatgggatt agacaccagaccatctataa aagaaagtgg gggaaaagaa gaaggacctc 1020 cacaggctta tcctattcaaacagtaaatg gagcaccaca gtatgtagcc cttgatccaa 1080 aaatggtgtc tatttttatggagaaggcaa gagaggggct aggaggtgaa gaagtccaac 1140 tgtggtttac agccttttcagctaatttaa catcaactga tatggctaca ttaattatgt 1200 ccgcacctgg ctgtgcagcagataaagaaa tcctagatga aacactgaaa cagatgacag 1260 ctgagtatga tcgtacccatcctcctgatg ggcctagacc gctgccctat ttcactgccg 1320 cagagatcat ggggataggattgactcaag aacaacaagc agaacccagg tttgccccag 1380 ccagaatgca gtgtagagcatggtatcttg aagcattagg aaagctagcg gccataaaag 1440 ccaaatctcc ccgagcagtacaattgaagc agggagctaa agaggactat tcctcattca 1500 tagatagact atttgctcaaatagatcaag agcagaacac agctgaggta aagctgtatt 1560 taaaacaatc tttgagcatagcaaatgcta atccagattg taagagagcg atgagtcatc 1620 ttaaaccaga aagtactttagaagagaaac tgagagcctg ccaggaaata ggatcgccag 1680 gatacaaaat gcaactattggcagaggctc ttactagggt gcaaacagtt caagcaaaag 1740 gaccaaggcc agtatgtttcaattgtaaaa aaccaggaca cctggccaga caatgtagac 1800 aagcaaagag atgtaataaatgtggaaaac ctggtcactt agctgctaac tgttggcaag 1860 gaggtaaaaa gtccccgggaaacggggcga tggggcgagc tgcagcccca gtaaatcaag 1920 tgcagcaagt gataccatctgcacccccgg tagaggagaa attgttagat atgtaaacta 1980 taataaagtg ggtaccaccacaactttaga aaaaagacct gaaatacaaa tattcgtaaa 2040 tgggtatcct ataaaatttttattagatac aggagcagat ataacaattt taaacagaaa 2100 agactttcag atagggaattctatagaaaa tgggaaacag aatatgattg gagtaggagg 2160 cggaaagaga ggaacaaattatatcaatgt gcatttagaa attagagatg aaaattataa 2220 gacacagtgt atatttggaaatgtgtgtgt cttggaggat aattcattaa tacaaccatt 2280 attgggaaga gataacatgattaagttcaa cataaggttg gtaatggctc aaatttcaga 2340 gaaaattcca atagtaaaagtaagaatgaa agaccctact caagggcctc aggtaaaaca 2400 atggccatta tcaaatgagaaaattgaagc tctaactgac atagtaaaca ggttagaaca 2460 agagggaaag gtaaaaagagctgatccaaa taatccttgg aacactcccg tatttgcaat 2520 caagaaaaag aatggtaaatggagaatgct catagatttt agggtcctaa ataaattaac 2580 agacaaaggg gcagaagttcagttaggact ccctcatcct gctggattac aattgaaaaa 2640 acaagtaact gtattggacataggggacgc atattttact attcctctag atccagatta 2700 tgctccttat actgcatttacactacctag aaaaaacaat gcaggaccag ggaggagata 2760 catatggtgt agtttaccacaagggtgggt cttgagtcca ttgatatatc agagtacctt 2820 agacaatata ctccaaccttttattaaaca gaatcctgag ttagatattt atcaatatat 2880 ggatgatatc tatataggatcaaatttaag taaaaaggaa cataaactaa aagtagaaga 2940 attaagaaaa ttgttattatggtggggatt tgaaaccccg gaagataaat tacaagaaga 3000 gcccccctat aagtggatgggctatgaatt acatccatta acgtggtcaa tacagcaaaa 3060 gcaattagaa attccagagagacccacatt aaatgaatta cagaagttag caggtaagat 3120 taactgggct agtcaaaccattccagactt gagcataaaa gaactaacta atatgatgag 3180 aggagatcaa aagttagactcaataagaga atggacgaca gaggccaaga atgaagtgga 3240 gaaagctaag agagcaattgagacacaggc acagctagga tattatgatc ctaatcgaga 3300 attatatgct aaattaagtcttgtgggacc acatcaacta agctatcagg tgtatcataa 3360 aaacccagaa cagatattatggtatgggaa aatgaatagg cagaagaaaa aagcagaaaa 3420 tacttgtgat atagctctaagggcatgtta caaaataaga gaagaatcca ttataagaat 3480 aggaaaagaa ccagtatatgaaatacctac atccagagaa gcttgggaat caaatctaat 3540 tagatctcca tatcttaaggcctcaccacc tgaggtggaa tttatacatg ctgccttaaa 3600 tataaaaaga gctctaagcatgatacaaga tgcccctata ttgggagcag aaacatggta 3660 catagatggg ggaagaaaacaaggaaaagc agcaagagca gcttattgga cagatacggg 3720 cagatggcag gtaatggaaatagaaggaag taatcaaaaa gcagaagtac aagctttatt 3780 attggcccta caggcaggaccagaggaaat gaatattata acagattcac aatatattgt 3840 gaatattatt aatcaacaaccagatttgat ggaaggaatt tggcaagaag tcttagaaga 3900 aatggaaaag aaagtagcaatctttataga ttgggtacct ggacataaag gtattccagg 3960 aaataaagag gtagatgaactttgtcaaac gatgatggtt atagaaggtg aaggaatatt 4020 agataaaaga tcagaagatgcaggatatga tttattagct gcacaagaaa tacatctctt 4080 gcctggggag gtaagagtagtaccaacaag aacaaagata atgttaccta aaggatattg 4140 gggattaata atgggaaaaagttcaatggg aagcaaagga ttagatgtat taggaggagt 4200 tatagatgaa ggatatagaggagaattagg ggtgataatg attaacctat ctaaaaaatc 4260 aataacatta tcagaaaaacaaaaagtagc acaattaata atattacctt gtaaacatga 4320 aagcttacaa caaggagaaataataatgga ttcagaaaga ggaagaaagg gatttgggtc 4380 aactggagtc ttttcttcatgggtggacag aattgaggaa gcagaattaa atcatgaaaa 4440 atttcactca gacccacaatacttaagaac agaatttaat ctacccagaa tagtagcaga 4500 ggaaataaaa agaaaatgtcccttatgtag aatcagaggg gaacaagtag ggggacaatt 4560 aaagattgga cctggcatatggcaaatgga ctgtacacac tttaatggaa aaataattat 4620 tgtcgcagtg catgtggaatcaggcttatt atgggcacag gtaattccac aggagactgc 4680 agattgtaca gttaaagctctcatgcaact tatcagtgct cataatgtta cagaactaca 4740 aacagataat ggaccaaattttaaaaatca gaaaatggaa ggactactaa attatatggg 4800 cataaaacac aaattaggtataccaggtaa cccacaatca caagcattag tagaaaatgc 4860 taaccacaca ttaaaatcttggattcaaaa atttctctca gaaacttctt ctttggacaa 4920 cgcattggcc ctagccttatactgcctcaa ttttaaacaa aggggtagac tagggagaat 4980 ggctccttat gaattatacatacaacagga atcattaaga atacaagact atttttcaca 5040 aattccacaa aaattaatgatgcaatgggt gtattataaa gatcagaaag ataaaaagtg 5100 gaagggacca atgagagtagaatattgggg acaaggatca gtattattaa agaatgaaga 5160 gaagggatat tttcttgtacctaggagaca cataagaaga gtcccagaac cctgcactct 5220 tcctgaaggg gatgagtgacgaagattggc aggtaagtag aagactcttt gcagttctcc 5280 aaggaggagt aaatagtgccatgttataca tatcgaattt acctgaaaca gaacaggcac 5340 aatataaaaa ggactttaagaaaaggctct tagaaaagga gactggattc atctatagat 5400 taagaaaagc tgaaggaataaggtggagct ttcatacgcg tgattattat ataggatatg 5460 taagagagat ggtggctgggtctagcctac aaaatagttt aagattgtat gtttatataa 5520 gcaatccatt gtggcatcagtcataccgtc ctggcctgac aaattttaat acagagtggc 5580 cttttgtaaa tatgtggataaagacaggat ttatgtggga tgatattgaa agccaaaata 5640 tttgcaaagg aggagagatctcacatggat ggggacctgg aatggtggga attgtgataa 5700 aagcatttag ctgtggagaaaggaagatac aaattactcc tgtcatgatt ataagaggtg 5760 agatagaccc acagaaatggtgtggagatt gttggaatct gatgtgtctt aaatattcac 5820 ttccaaatac attgcagaggcttgctatgc tggcgtgtgg caaagaggct aaagaatgga 5880 gaggctgttg taatcagcgttttgtttctc ctttcagaac accctgtgat ctagaggtcg 5940 tccagaacaa gcctaaaaggaatttattgt ggacgggaga attatgaatg gaagaaataa 6000 tcccactgtt taataaggttacagaaaagt tagatagaga agcagctatt agattgttta 6060 ttttagctta tcaggtagacagatgcagat ttattagaat tttacaatta ttactttgga 6120 gagatagatt taagtcaatcaattctaaat attgtttatg ctggctgtgc tgcaagtctg 6180 cttattggcg cttgcaatctacattatcca taaatactgc ctagaaatat ttcttttaat 6240 atttcatctg cagatataaacatggcagag ggaggattta ctcaaaatca acaatggata 6300 gggccagaag aagctgaagaattgttagat tttgatatag ctgtacaaat gaatgaagaa 6360 ggtccattaa acccaggagtaaacccattt agggtaccag gaattacctc tcaagaaaag 6420 gatgattatt gtcagattttacaaccaaaa ctacaagaat taaagaatga aatcaaagag 6480 gtaaaacttg acgaaaacaatgcaggtaag tttagaaagg caagatattt aagatattct 6540 gatgagagtg tactaactatagtctattta ctaacaggat atttgagata tttaataagc 6600 catagaaact taggatctttaagacatgat atagatatag aagcaccaca acaagagcac 6660 tataatgata aagaaaagggtactacttta aatataaagt atgggagaag atgttgtatt 6720 agcacattac ttctatatttaatcctcttc tcagggatag gaatttggct tggaaccaaa 6780 gcacaagtag tgtggagactccctccttta gtagtgccag tagatgagac agaaataata 6840 ttttgggatt gttgggcgccagaggaacca gcctgtcaag attttctggg aacaatgata 6900 catttaaaag caaatgttaatataagtata caagaaggac ctacattggg aaattgggca 6960 agggaaattt ggtctacattatttaaaaaa gctacaaggc aatgcagaag gggaaggata 7020 tggaagaaat ggaatgagactataacagga cctaaaggat gtgcaaataa tacctgttat 7080 aatatttcag tagtggtacctgattatcaa tgttatgtag acagagtaga tacatggctg 7140 caaggaaaag ttaatatctcactatgtttg acaggaggaa agatgctata taataaaaat 7200 acaaaacaat taagttactgtacagatcca ttacaaatac cattaattaa ttacacattt 7260 ggacctaacc aaacttgtatgtggaacaca tctttaatca aagaccctga gataccgaaa 7320 tgtggatggt ggaaccaggcagcctattat aataattgta aatgggaaga agctaatgtg 7380 acatttcaat gtcaaagatcacaaagtcta ccaggatcat gggttaggag aatctcttca 7440 tggagacaaa gaaacagatgggagtggagg ccagactttg aaagtgagaa agtaaaaata 7500 tcattacaat gtaatagtacaaaaaattta acttttgcaa tgagaagttc aagtgattat 7560 tatgatgtac aaggagcatggatagaattt ggatgttata gaaataaatc aagaacccat 7620 acgggagcaa gatttagaataagatgtaaa tggaatgaag gaaagaatct atctctcatt 7680 gatacatgtg ggactacttcaaatgtgaca ggagccaacc ctgtagattg tactatgaaa 7740 acaagcacta tgtacaattgttccttacaa gatagtttca ctatgaaaat agaggacctt 7800 attgtacaat ttaatatgacaaaagcagtg gaaatgtata atattgctgg gaattggtct 7860 tgtacatctg atttaccaacagggtgggga tatatgaaat gtaattgtac aaatgccact 7920 gatggggaga ataaaatgaaatgccctagg aatcagggta ttttaagaaa ctggtacaat 7980 ccagttgcag gactaagacaagctcttatg aagtatcaag tagtaaaaca accagaatat 8040 ttggtggtac cggaagaagttatgaggtat aaaggtaaac aaaaaagggc cgctattcat 8100 attatgttag cccttgctacggtgttatct atagctggag caggaaccgg tgccactgct 8160 attgggatgg tgacacactatcagcaagtt ttggctaccc atcagcaggc attggacaaa 8220 ataactgagg cactgaaaataaacaactta aggttaatca ctttagaaca tcaagtatta 8280 gtgatagggt taaaagtagaggctatagaa aaattcctat atacagcttt tgctatgcaa 8340 gaattaggat gtaatcagaatcaattcttt tgtaagattc ccctcaatct gtggacaatg 8400 tataacatga ctataaatcatacactatgg aatcatggaa atataacttt gggagaatgg 8460 tataatcaaa caaaaagtttacaagaaaaa ttttatgaga taattatgga tatagaacaa 8520 aataatgtac aagggaaaaatggaatacaa caattacaaa aatgggaaaa ttgggtggga 8580 tggataggca aaatccctcaatatttaaaa ggacttcttg gtagtgtgtt gggaatagga 8640 ctaggaatct tactactacttatatgcttg cctacattag tagattgtat aagaaactgt 8700 actaataaaa tattgggatatacagttatt gcaatgcctg aaatagatga tgaggaagta 8760 cacccatcag tggaattgaggagaaatggc aggcaatgtg gcatatctga aaaagaggag 8820 gaatgatgga gcatttcagacctgtagaat acaggagtaa tgctgagctg agttcttccc 8880 tttgaggagg atgtgtcatatgaatccatt tcaaatcaaa aataacagta aaatctatat 8940 tgtaaggcaa acgaaaaagacaacgcagaa gaagaaagaa gaaggccttc aaaaaattga 9000 tgctggattt agaggctcgatttaaagcgt tgtttgaaac accttcagct acagaatata 9060 ctgcagacga gacagaagaagagactcttg aaaaagaaaa aagggtggac tgggaagatt 9120 attgggatcc tgaagaaatagaaaaaatgc taatggactg aggacgtaca taaacaagtg 9180 acagatggaa acagctgaatatgactcaat gctagcagct gcttaaccgc aaaaccacat 9240 cctatgtaaa gcttgccgatgacgtgtatc ttgctccatt ataagagtat ataaccagtg 9300 ttttgtaaaa gcttcgaggagtctctctgt tgagggcttt cgagttctcc cttgaggctc 9360 ccacagatac aataaaaaactgagctttga gattgaaccc tgtcttgtat ctgtgtaatt 9420 tctcttacct gcgaatccctggagtccggg ccagggacct cgca 9464 2 30 DNA Artificial Sequence Descriptionof Artificial Sequence PRIMER 2 ccgcaaaacc acatcctatg taaagcttgc 30 3 30DNA Artificial Sequence Description of Artificial Sequence PRIMER 3cgcccctgtc cattccccat gttgctgtag 30 4 30 DNA Artificial SequenceDescription of Artificial Sequence PRIMER 4 acaaacagat aatggaccaaattttaaaaa 30 5 30 DNA Artificial Sequence Description of ArtificialSequence PRIMER 5 gcaatgtggc atgtctgaaa aagaggagga 30 6 26 DNAArtificial Sequence Description of Artificial Sequence PRIMER 6tctgtgggag cctcaaggga gaactc 26 7 34 DNA Artificial Sequence Descriptionof Artificial Sequence PRIMER 7 tcttcccttt gaggaagata tgtcatatga atcc 348 30 DNA Artificial Sequence Description of Artificial Sequence PRIMER 8ttactgtttg aataggatat gcctgtggag 30 9 30 DNA Artificial SequenceDescription of Artificial Sequence PRIMER 9 ttaaaggatg aagagaagggatattttctt 30 10 28 DNA Artificial Sequence Description of ArtificialSequence PRIMER 10 tttcaatatc atcccacata aatcctgt 28 11 30 DNAArtificial Sequence Description of Artificial Sequence PRIMER 11tgggaagatt attgggatcc tgaagaaata 30 12 40 DNA Artificial SequenceDescription of Artificial Sequence PRIMER 12 catatcctat ataataatcacgcgtatgaa agctccacct 40 13 40 DNA Artificial Sequence Description ofArtificial Sequence PRIMER 13 aggtggagct ttcatacgcg tgattattatataggatatg 40 14 24 DNA Artificial Sequence Description of ArtificialSequence PRIMER 14 tgcgaggtcc ctggcccgga ctcc 24 15 29 DNA ArtificialSequence Description of Artificial Sequence PRIMER 15 ctccagggattcgcaggtaa gagaaatta 29 16 34 DNA Artificial Sequence Description ofArtificial Sequence PRIMER 16 tttcatctgc atcgataaac atggcggagg gagg 3417 33 DNA Artificial Sequence Description of Artificial Sequence PRIMER17 cctgtattct actagtctga aatgctccat cat 33 18 34 DNA Artificial SequenceDescription of Artificial Sequence PRIMER 18 tttcatctgc atcgataaacatggcggagg gagg 34 19 49 DNA Artificial Sequence Description ofArtificial Sequence PRIMER 19 gggctaacat aatatgacta gttcaccttttttgtttacc tttatacct 49 20 34 DNA Artificial Sequence Description ofArtificial Sequence PRIMER 20 ggtaggatcg attctacagc aacatgggga atgg 3421 42 DNA Artificial Sequence Description of Artificial Sequence PRIMER21 gtcttcacta gtaagttgtg gtagtaccca ttgtattata gt 42 22 38 DNAArtificial Sequence Description of Artificial Sequence PRIMER 22ggtaaaaatc gatcatgaaa cggggcgatg gggcgagc 38 23 36 DNA ArtificialSequence Description of Artificial Sequence PRIMER 23 actgcaactagtcttctact tacctgccaa tcttcg 36 24 37 DNA Artificial SequenceDescription of Artificial Sequence PRIMER 24 tgttagatcg ataatgtataataaagtggg taccacc 37 25 36 DNA Artificial Sequence Description ofArtificial Sequence PRIMER 25 actgcaacta gtcttctact tacctgccaa tcttcg 3626 34 DNA Artificial Sequence Description of Artificial Sequence PRIMER26 ggtaggatcg attctacagc aacatgggga atgg 34 27 37 DNA ArtificialSequence Description of Artificial Sequence PRIMER 27 ctgtttggggcccataagcc tgtggaggtc cttcttc 37 28 44 DNA Artificial SequenceDescription of Artificial Sequence PRIMER 28 ggacctatcg ataccatgcctattcaaaca gtaaatggag cacc 44 29 36 DNA Artificial Sequence Descriptionof Artificial Sequence PRIMER 29 tgcttggggc ccttgcaccc tagtaagagc ctctgc36 30 42 DNA Artificial Sequence Description of Artificial SequencePRIMER 30 ggctcttatc gataccatga cagttcaagc aaaaggacca ag 42 31 38 DNAArtificial Sequence Description of Artificial Sequence PRIMER 31tattatgggc cccatatcta acaatttctc ctctaccg 38 32 44 DNA ArtificialSequence Description of Artificial Sequence PRIMER 32 ccctgcactcttcatcgata ccatgagtga cgaagattgg cagg 44 33 47 DNA Artificial SequenceDescription of Artificial Sequence PRIMER 33 gggattattt cttcgggccctaattctcct gtccacaata aattcct 47 34 42 DNA Artificial SequenceDescription of Artificial Sequence PRIMER 34 ttgtggacgg gaatcgataccatggaagaa ataatcccac tg 42 35 42 DNA Artificial Sequence Description ofArtificial Sequence PRIMER 35 atattaaaag aaatagggcc cggcagtatttatggataat gt 42 36 46 DNA Artificial Sequence Description of ArtificialSequence PRIMER 36 gtataaaggt atcgatacca tggccgctat tcatattatg ttagcc 4637 42 DNA Artificial Sequence Description of Artificial Sequence PRIMER37 tgaaatgctg ggcccttcct cctctttttc agatatgcca ca 42 38 34 DNAArtificial Sequence Description of Artificial Sequence PRIMER 38tttcatctgc atcgataaac atggcggagg gagg 34 39 36 DNA Artificial SequenceDescription of Artificial Sequence PRIMER 39 tgtacggggc ccgtccattagcattttttc tatttc 36 40 37 DNA Artificial Sequence Description ofArtificial Sequence PRIMER 40 tgttagatcg ataatgtata ataaagtggg taccacc37 41 43 DNA Artificial Sequence Description of Artificial SequencePRIMER 41 ctctgaaacg ggccccatta ccaaccttat gttgaactta atc 43 42 46 DNAArtificial Sequence Description of Artificial Sequence PRIMER 42aacataatcg ataccatggt ccagatttca gagaaaattc caatag 46 43 39 DNAArtificial Sequence Description of Artificial Sequence PRIMER 43ttctaggggc cccatcgttt gacaaagttc atctacctc 39 44 43 DNA ArtificialSequence Description of Artificial Sequence PRIMER 44 ctttgtatcgataccatggt tatagaaggt gaaggaatat tag 43 45 37 DNA Artificial SequenceDescription of Artificial Sequence PRIMER 45 cacccagggc ccaaagactccagttgaccc aaatccc 37 46 42 DNA Artificial Sequence Description ofArtificial Sequence PRIMER 46 gggtcaatcg atacaatgtc ttcatgggtggacagaattg aa 42 47 38 DNA Artificial Sequence Description of ArtificialSequence PRIMER 47 caatctgggc ccctcatcac cttcaggaag agtgcagg 38 48 32DNA Artificial Sequence Description of Artificial Sequence PRIMER 48tgtagagcat ggtatcttga agcattagga aa 32 49 35 DNA Artificial SequenceDescription of Artificial Sequence PRIMER 49 gttcctctct ttccgcctcctactccaatc atatt 35 50 113 DNA Artificial Sequence Description ofArtificial SequenceMultiple Cloning Sites 50 gcggccgcaa gatatcgccctaggtaagat ctcgatcgat ttggtaccaa tcgcgacctt 60 aattaacagc tagcggatttaaatcagggc ccgggatact agtgagcggc cgc 113 51 108 DNA Artificial SequenceDescription of Artificial SequenceMultiple Cloning Sites 51 gcggccgcaagatatcgccc taggtaagat ctcgatcgat ttggtaccaa tcgcgacctt 60 aattaacagctagcggattt aaatcagggc ccactagtga gcggccgc 108 52 42 DNA ArtificialSequence Description of Artificial SequenceEpitope Tag and Stop Codon 52atgcagtacc cctacgacgt ccccgactac gccatgcatt ga 42

What is claimed is:
 1. An isolated antibody that binds specifically toan FIV protein.